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The effects of postoperative time intervals and diurnal variation on liver glycogen levels in rats with hippocampal lesions
Pergamon Press
Life Sciences, Vol . 17, pp . 1607-1616 Printed in U .S .A .
'f l ~ ~FLCTS OF P(.,`5TC7'EP~ATIV :I ^lIt'JD II~JTERVALS AIiU DIURNAL VARIATIG I ON LIVr:R fLYC()~ï~:Iv LFVFhS IPJ nATS :"JI17i Iill'Pï,CAJiPAL 1~ESIOEJS Cyrilla fI . lJi.deman, John Carroll University, Cleveland, Ohio
4411
Helen ;
44118
Thomas S, Brown, DePaul University, Chicago, Illinois
60614
(Received in final form October 27, 1975) Summary Liver glycogen levels were measured in rats with hippocampal lesions and in control animals . Liver glycogen levels were determined each week for the first ten postoperative weeks and eight months postoperatively . It was found that after one week, control animals and animals with hippocampal lesions were not significantly different in liver glycogen levels . By the end of the second week the group with hippocampal lesions was significantly higher than the control animals . This variation pattern continued during the third week . By the end of the third week the animals with hippocampal ablations had reached their highest level, which remained unchanged throughout the rest of the series . No significant changes in liver glycogen levels were obtained in the control animals . Liver glycogen levels were then measured in normal rats and rats with hippocampal lesions maintained on a diurnal rhythm of twelve light hours followed by twelve dark hours . One month postoperatively, rats with hippocampal lesions had a significantly higher liver glycogen level at all time periods as compared with normal animals . Both groups of rats showed the diurnal pattern of higher levels of liver glycogen in the beginning of the light phase and lower levels of liver glycogen in the beginning of the dark phase . The observed variations may be explained in terms of alterations in known homeostatic mechanisms controlling liver glycogen levels . Currently there is an interest in the study of the interrelationship between the hippocampas and the hypothalamic-pituitary-adrenal system . The inhibitôry influence of the hippocampas upon the pituitary-adrenocortical system is well documented . In 1961 Fendler, Karmos and Telegdy (1) reported that extensive lesions of the hippocampas of the cat caused a three-fold increase in secretion of glucocorticoids . Knigge (2~ and Knigge and Hays (3) reported significant increases in basal plasma corticoid levels following bilateral electrolytic hippocampal lesions in rats . Endroczi, Lissak, Bohus and Kovacs (4) and Moberg, Scapagnini, DeGroot and Ganong (5) have domonstrated an inhibitory influence upon the pituitaryadrenocortical system with stimulation of the hippocampas . Mc~wen, ldeiss and Schwartz (6, 7) have shown that radioactive corticosterone injected into rats is taken up by all parts of the brain, but there is a tendency 1607
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for the hippocampus and septum to concentrate and retain the labeled corticosterone . The second study of these authors (7) showed that nuclear retention of the labeled hormone was found to be highest in the hippocampus . In 1972 Gerlach and P"IcEwen (8) showed that when tritiated corticosterone is injected subcutaneously into adrenalectomized male rats one hour before killing, intense labeling of the hippocampus is produced . Glucocorticoids have been shown to have various effects upon protein, carbohydrate and lipid metabolism . Notable among these effects is an increase in enzyme production, especially those enzymes responsible for gluconeogenesis (9, 10, 11, 12) . Weber, Banerjee and Bronstein (13) have shown that cortisone administration increases selectively certain liver enzymes involved in gluconeogenesis, whereas other enzyme systems are not affected, Gleber, Singhal and Stamm (14) have reported that administration of cortisone to rats causes marked de novo synthesis of glucose-6phosphata,se, fructose-l,6-diphosphatase, aldolase and lactic dehydrogenase, all of which are key enzymes in the process of gluconeogenesis . Another important effect of the glucocorticoids is to direct amino acids from tissues to the liver for deamination (15) . In addition, the glucocorticoids have an inhibitory effect on lipogenesis which appears to be of secondary origin and is possibly related to the gluconeogenesis and marked reversal of glycolytic reactions which supply substrates for fat synthesis (15, 16) . The increase in deaminated amino acid residues as well as oxidation fragments (15) entering the glycolytic pathway and Krebs cycle, as well as increases in enzymes concerned with gluconeogenesis, all directly or indirectly due to increased levels of glucocorticoids, would cause a shift in chemical equilibrium resulting in gluconeogenesis . Sie and Fishman (17) have reported that glucocorticoids stimulate glycogen synthetase activity, an enzyme responsible for glycogen synthesis . Thus increased gluconeogenesis and glycogen synthetase activity would result in increased liver glycogen levels with increased circulating glucocorticoids . I'D should be mentioned here that increased levels of glucose-6phosphatase seen with increased levels of circulating glucocorticoids (14) could result in increased blood glucose levels . Murphy, Wideman and Brown (18) did not find an increase in blood glucose levels in animals with hippocampal lesions . In the same study, however, these authors showed that rats with hippocampal lesions had higher liver glycogen concentrations than normal and cortical control animals . The effect of diurnal variation upon liver glycogen levels in rats with hippocampal lesions has not yet been determined . In 1959 Halberg, Peterson and Silber (19) observed a 24-hour rhythm (diurnal variation on a 12 hr . light - 12 hr, dark schedule) of glucocorticoids in mouse serum . The highest level was observed in the eleventh hour of the light period . Similarly, Zimmermann and Critchlow (20) reported that in normal rats there is a diurnal fluctuation in plasma levels of corticosterone with the highest levels being reached in the late afternoon . Their lighting schedule consisted of 14 hrs . of light (from 4c00 a .m . to 6s00 p .m .) followed by 10 hrs, of darkness . In 1966 Slusher (21) implanted cortisol in the ventral hippocampus with a resultant loss in diurnal variation of plasma corticosteroids . Recent data concerning the effects of sectioning the fornix, the major efferent pa~ttsway from the hippocampus, and its influence on plasma corticosterone levels are
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Liver Glycogen and Hippocampal Lesions
Whereas Moberg, Scapagnini, DeGroot and Ganong (5) found contradictory . that sectioning of the fornix abolished the diurnal variation in plasma corticosterone levels, Wilson and Critchlow (22) reported that fornix transections were compatible with normal steroid patterns in individual rats . From some of the above studies (1, 2, 3, 4) it has been domonstrated that there are alternations in glucocorticoid levels when the hi pocampus is removed or stimulated . In addition, sectioning the fornix (5~ may These manipulations bring about alterations in glucocorticoid levels . should then have an effect upon both the level and diurnal variation of liver glycogen levels . The present study, therefore, has two purposes : 1) to examine the time course of the increase in liver glycogen levels following ablation of the hippocampus, and 2) to study the effect of 3iurnal variation upon liver glycogen levels in rats with hippocampul lesions. Experiment 1 -_Method Animals The animals used were 114 male Sprague-Dawley rats weighing between 245 and 280 grams at the time of operation. The animals were weigJzed and randomly divided into three groups o£ 38 animals each . The first group received bilateral hippocampul lesions (H) . The second group served as cortical controls (C) and the third group served as normal controls (N) . Bilateral lesions o£ the hippocampus were made as completely as possible by aspirating both medially and ventrally. The overlying cortex was removed on both sides . In cortical control animals only the cortex overlying the hippocampus was removed on both sides by aspiration . Histological Procedure The animals were sacrificed between 8 :00 and 9130 a.m . by decapitation . The brains were immediately removed and placed in lOJ formalin . The brains were embedded in paraffin and sectioned at 15 xt . Every tenth section through the lesion was mounted and stained according to the method o£ Klüver and Barrera (23) " Hippocampal and cortical control lesions were similar to those reported by i~furphy and Brown (24), Extent of damage to the hippocampus ranged from about 70l to 90~ of the structure with most of the ventral tip usually being spared . Experimental Proçedure Animals had ad-lib . access to food and water throughout the experiment and were placed in alternating periods of light and darkness each The experi day. The lights went on at 8:00 a .m . and off at 8s00 p.m . ment was run each week £or the first ten postoperative weeks and eight months postoperatively . For the first five weeks of the experiment four animals from each group were sacrificed at the specified time . For the second five weeks of the experiment as well as for the eight month time period, three animals from each group were sacrificed at the specified time . After decapitation the animals were opened on the ventral side and the liver was removed and placed in a petri dish on ice. The entire liver was weighed and two separate 100 mg . samples were removed from the peripheral third o£ the liver. The liver samples were ground with 10 ml . of 5J trichloroacetic acid in a homogenizer. The homogenate was placed in a The centrifuged homogenate was anatest tube and centrifuged for 5 mir.. lyzed for glycogen according to the iodine method (25) .
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Experiment 1 - Results and Discussion The results of experiment 1 are summarized in Figure 1 . After one week the three groups did not differ significantly in liver glycogen
F
M 7 . W
0 V
F 6
O---O ~"
Normal animal: Hippocompal lodonod animals Q-d Cortical animals
TIME
FIG . 1 Relationship between glycogen concentration and time following lesions . levels . By the end of the second week animals with hippocampal lesions were significantly higher than the control groups . Dy the end of the third week animals with hippocampal ablations had reached their highest level of liver glycogen, This high level remained unchanged throughout the rest of the experiment . Pio significant differences in liver glycogen levels were observed in the control animals . It can be noted that the liver glycogen levels obtained eight months posto eratively replicate the earlier findings of tiurphy, Wideman and Brown (18~ that animals with hippocampal lesions have significantly higher liver glycogen levels than control animals at this time period . Table 1 gives the glycogen concentration in mg . of glycogen per g . of liver for each group at the various time intervals . The numbers have been derived from a standard curve for glycogen concentration . The Mannhihitney U Test showed that animals with bilateral hippocampal lesions ha.d significantly higher levels of liver glycogen than control animals from the second week on . For the second through fifth weeks : H vs . C, U = 0, p = .014 and H vs . N, U = 0, p = .014 . For the sixth throw the tenth weeks and for eight months : H vs . C, U = 0, p = .Oj and Ii vs . N, U = 0, p = .Oj . The animals with hippocampal lesions usually had over twice the amount of liver glycogen as did the other animals from three weeks, until the end of the experiment at eight months . The fact that it took three weeks for this phenomenon to develop indicates that there are biochemical changes occurring in the animals with hippocampal ablations during this time period . From these findings it would appear that such changes should be kept in mind when conducting behavioral studies involving animals with
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161 1
hippocampal lesions . In other words, it is suggested that behavioral studies be conducted after these biochemical changes have stabilized . TABLE 1 Summary of ßesults of Liver Glycogen and Postoperative Time Intervals Postop . Group
mg Glycogen per g of Liver
Postop .
Group
mg Glycogen per g of Liver
1 wk .
H G N
322 .5 287 .5 275 .0
7 wks .
II G N
535 " 0 z55 " 0 270 .0
2 wks .
H C N
500 .0 282 .5 250 .0
8 wks .
H G N
570 .0 255.0 275 .0
3 wks .
x C N
650 .0 282 .5 255 .0
9 wks .
x G N
610 .0 287 .5 270 .0
4 wks .
H C N
610 .0 265 .0 275 .o
10 wks .
H C N
510 .0 260 .0 292 .5
5 wks .
H G N
625 .0 285 .5 z5o,o
8 mos .
H c N
610 .0 237 .5 z8z .5
6 wks .
H C N
610 .0 z3o .o 265 .0
Many factors affect the level of liver glycogen in an animal . One factor is food intake of such animals . Boitano, Lubas, Auer and Fernald (26) have shown that rats with hippocampal lesions do not increase food intake . Therefore, the increase in liver glycogen is not brought about by an increase in food intake . A second factor is diurnal variation . Basically, this involves the light-dark cycle . This factor will be considered in experiment 2 . In many cases the two factors overlap in the ra.t . IJith the onset of the dark period the nocturnal rat eats considerably more than in the light cycle . In the present experiment the above two factors were held constant . The animals were fed ad-lib . and were maintained on a 12 hr . light - 12 hr, dark cycle . A third factor is the level of glucocorticoids . Previous studies such as those of Fendler, Karmos and Telegdy (1), Knigge (2) and ICnigge and Hays (3) have shown that the level of glucocorticoids increases in hippocampally-damaged animals . Although Coover, Goldman and Levine (27) did not find an increase of glucocorticoids in animals with hippocampal lesions, their measurement of glucocorticoids followed behavioral experiments which involved water deprivation . Sakellaris and Vernikos-Danellis (28) suggest caution in characterizing animals as normal or adapted in experiments in which some type of stressful regimen such as water deprivation is employed . In the present experiment no stress was introduced into the experimental design . The glucocorticoids have an influence on gluconeogenesis . '~lith an increased level of glucocorticoids the process of gluconeogenesis is speeded up . In addition to gluconeogenesis, the
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glucocorticoids also affect the ability of the liver to store glycogen With an increase of these two activities of the glucocorticoids, (29) . liver glycogen levels should be elevated . Murphy, ~lideman and Brown (18) have shown that liver glycogen levels are significantly higher in rats with hippocampal ablations eight months postoperatively . The present study indicates that increased liver glycogen storage gradually develops over a three week time period in rats with hippocampal lesions . Furthermore, this phenomenon appears to be long lasting rather than temporary. Experiment 2 - Method Animals The animals used were 48 male Sprague-Dawley rats weighing between The animals were weighed and randomly divided into two groups of 24 animals each . The first group received bilateral hippocampal lesions and the second group served as normal controls . A cortical control group was not utilized since previous studies (1£3, experiment 1 of the present study showed that normal and cortical control animals did not differ significantly . Bilateral lesions of the hippocampas were made as completely as possible by aspirating both medially and ventrally . The overlying cortex was removed on both sides.
215 and 245 grams at the onset of the experiment .
Histological and Experimental Procedures The basic histological and experimental procedures were the same as those described in experiment 1 with the following exceptions, The experiment was run one month postoperatively, Three animals from each group were sacrificed at 9 a,m noon, 3, 6, 9 p,m ., midnight, 3 and 6 a,m . Experiment 2 - Results and Discussion A comparison of operated animals and normal animals with respect to liver glycogen levels at various intervals of time is shown in Figure 2, At 9 a .m . the highest liver glycogen level was reached . By 9 p,m. the greatest amount of glycogen had been mobilized from the liver. Although both normal animals and animals with hippocampal lesions maintained diurnal variation in liver glycogen levels, it is apparent from Figure 2 that the latter group possessed markedly higher liver glycogen levels . Within each time interval there were no overlaps of individual animals in the operated group with animals in the normal group. The t4ann-Whitney U Test for each time interval showed that the differences were significant (U = 0, p = ,05) . It is also interesting to note that the general pattern of the two graphs could be readily superimposed upon each other without noticeable variations . Thus both animal groups appear to possess a regulating system which gradually increases or decreases the liver glycogen level . The actual mg, of glycogen per ml . of test solution can be derived from the standard curve of glycogen shown in Figure 3 . On the standard curve only the peak and trough points have been plotted . At 9 a.m . normal animals maintained a glycogen level of 1 .46 mg~ml of test solution . Animals with hippocampal lesions showed a significant increase of liver glycogen at the same time period . The animals reached a peak of 2.24 mg~ml of test solution . Conversely, at 9 p.m . normal animals showed a decrease in liver glycogen to 0,2 mg~ml of test solution . Animals with hippocampal lesions also showed a decrease at 9 p .m but still had a
Vol . 17, No . 10
Liver Glycogen and Hippocampal Lesions
55r 50 45 40 N .35 z ° .30
20 15
.la 05
Pt .05
n 9 om
0
12 noon
n 3 pm
o 6 pm
n n 9 12 pm midn,
n 8 am
3 am
9 am
TIME
FIG .
2
A comparison of hippacampal lesioned and normal animals with respect to diurnal variation in liver glycogen levels . STANDARD CURVE FOR GLYCOGEN
60 .50
O Na~ma1 animale " Hipposampal lesioned animals
a .40 i ° .30 20
t
4
~
B
v
1.2
~
L&
~
2.0
mp GLYCOCaENfmITE51 SOLUTION
_
~2 .4
FIG,
Standard evsve of glycogen concentration showing Beak and trough points of hippocampal lesioned and normal animals.
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significantly higher level of liver glycogen than normal animals, namely, 0 .5 mg~ml of test solution . Values for all the time periods taken from the standard curve are given in Table 2 . TA}3LE 2 Summary of Results of Liver Glycogen during the Diurnal Cycle Time
Group
mg Glycogen per g of Liver
Time
Group
mg Glycogen per g of Liver
9 a .m .
H N
560 .0 365 .0
9 P" m.
x ra
125 .0 50 .0
12 noon
H N
455 .0 335 " o
12 midn .
H N
180 .0 120 .0
3 P .m .
H r7
265 .0 182 .0
3 a .m .
H N
210 .0 180 .0
6 p .m,
H N
220 .0
6 a .m .
H
380 .0 2?5 .0
loo.o
rr
The data presented in this experiment clearly show the effect of diurnal variation on liver glycogen levels, which was not considered in the earlier work of IWrphy, ldideman and Brown (18) . These observations lead one to speculate on the role of the glucocorticoids in the phenomenon of diurnal variation and ultimately the important regulatory part played by the hippocampus . Halberg, Peterson and Silber (19) have reported that the glucocorticoids reach their peak level in the eleventh hour of the light period . In the present experiment this time period corresponds to 7 p .m ., - one hour before the lights go off and before the rat enters into its activity phase . According to Thompson and Lippman (30), the currently popular model for glucocorticoid action states that there is an altered rate of synthesis of certain species of RNA . itibosomal and transfer RNA synthesis are altered depending on the tissue, and presumably there are quantitative and possibly qualitative alterations in the rate of messenger (mRNA) synthesis . These alterations are reflected in altered rates of synthesis of specific proteins (enzymes) . It appears to be established that in most carefully studied examples of enzyme induction by steroids there is a quiet period, or lag, after the hormone reaches the cells, before increased enzyme activity or functional mRNA can be observed (31) " Shortly after the limits o off at 8 .m ., the animal begins to eat, thus increasing the substrate amino acids necessary for gluconeogenic activity . This feeding behavior continues until the lights go on at 8 a .m . Since the level of glucocorticoids is highest one hour before the onset of the dark period, the production of liver gluconeogenic enzymes would slowly increase in quantity . As the digestive process continues in the gut of the rat after eating, the concentration of amino acids increases . ldith increased quantities of amino acids being removed by the liver, and increased quantities of liver gluconeogenic enzymes being produced, the rate of gluconeogenesis would be increased, conceivably reaching its maximum point around 9 a .m ., one hour after the rat has ceased its activity phase . At this point, the glycogen level has peaked . Since the hippocampectomized rat has a diurnal variation pattern similar to ttie normal animal, but in all cases with higher levels of liver glycogen, one could postulate a regulatory role by the hippocampus on
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Liver Glycogen and Hippocampal Lesions
1615
glucocorticoid secretion . This regulation could possibly be via the hypothalamus since Nauta (32) has reported neuronal connections between the hippocampus and the arcuate nucleus of the hypothalamus in the rat . In summary, it has been shown that diurnal variation affects liver glycogen levels in rats . Although removal of the hippocampus does not alter the diurnal cycle, it appears to have an influence upon the amount of glycogen stored by the liver . Acknowledgment - Supported in part by a Research Grant from the Dept . of Mental Health of the State of Illinois to Thomas S . Brown . Ref erences 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17, 18 . 19 . 20 . 21 . 22 . 23 . 24 . 2$ . 26 .
K, r^EIIDLE3, G . I(ARI"iGS and G . TELr7GDY, Açta Physiol . Açad . Scient . ~ . 20 293-297 (1961) . K . I~Y . IOïIGGE, Proc . Soc . E~ . Biol . hied . 108 18-21 (1961) . h . I'i . IQ3IGGE and I"'i . I-iAYS, Proc . Soc . E~ . Biol . I"Yed . 114 67-68 (1963) " E . ENDRO(:ZI, K . LISSAK, E . BCHiUS and S . KOVAC5, Açta Phvsiol . Açad . Scient . h~ . 16 17-22 (1959) " G . P . MOBFG, Ü. SCAFAGIdI:~I, J . DJGRGOT and 4 . F . GAI~IONG, Idearo endocrinolo~y ~ 11-15 (1971) . B . S . IwiCE:"IfP ;, J . I%i . 'riEISS and L, S . SCHIIAR'T'Z, Lrain i?eso 16 227-241 (1969) . E . S . P4CETIEN, J . I^ . ~~ISS and L . S . SCH ïAÜ1"L, Brain Res . 471-482 (1970) . J . L . GERLACH and B . 5 . I"ICE~IEId, Science ~ 1133-1136 (1972) . E . SHIRAGC, H . A . LARDY, R . C . IdORDLIE and D . 0 . FOSTER, J . Biol . Chem . ~ 3188-31g2 (1963) . G . 17EBER, S . K . SRIVASTAVA and R . L . SINGHAL, J . Biol . Chern . 240 750-756 (1965) " I{ . V . HEIIîiIIdG, id . IfUTrI and I . SEiTBERT, Eiochem . Bio s . Research Communs . ~ 496-501 (1964) . R . J . I4ARTIN, J . A . PATT and R . J . i~BERIiART, Proc . Soc . ~ . Biol . G . I:~~:a;;, G . BAP+c.dJEE and 5 . B . Bï20IiSTEIAI, J . Biol . Chem . 2 ;6 3106-3111 (1961) . G . LdEBra, R . L . SIiJGIIAL and :~' . B . STAMM, Science _l42 390-392 (1963) . ~ . S . >EST, 'r1 . R . TGDD, II . S . I`ïASOIv and J . ^t . VAI B3UGGDri, Textbook of Biochemistry , The I~iacmillan Company, venr York (1966) . R. H . VIILLIAIIS (ed .)s Diabetes , Paul B . Hoeber, Inc ., New York (1960) . H . SIE and ïl . H . FISHI"ïAld, Science 1~ 816-817 (1964) . H . II . i~NRPHY, C . H . 1IDr~4AId and T . S . BRO+:I ;, Phvsiol . Eehav . £; 1171-1174 (1972) . F . i-iALBEPG, i? . E . PETERSGl"1 and R . II . SILi;r.Ft, Endocrinolo~.y ä+ z22-230 (1959) . E . ZTi"uri~?AI1I1 and V . CRiTCIILUIT, _Am . _J . Ph siol . _216 148-155 (1969) . I"I . A . SLUSïIER, ~ . Brain Res . 1 184-194 19 ) . 'i~I . ~dIISOTd and V . CRITCHLO'r1, iveuroendocrinology ~ 29-40 (1974) . H . IIZUVER and E . BARFRA, J . Neuropath . ~ . Neurol . 12 400-403 (1953) " H . i'I . i"IURPHY and T . S . BFtOV1id, J . Comp . Phvsiol . Psychol . Z 404-415 (1970) " J . VAN DER VIES, Biochem , J . ~ 410-416 (1954) . J . J . BOTTANO, J . F . LUBAR, J . AUER and M . S . FERNALD, Physiol . Behau . _3 901-906 (1968) .
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Liver Glycogen and Hippocampal Lesions
27,
G . D. CUUV;JR, L . GULDi:AiQ and S . LEVII~~, i'h:~siol . Behav . ~
28 .
P . C . SAi~LL.~IS and J . V~~idIKUS-DAI~c LLIS, Physiol, i3ehav ,
29 .
V . D, i~IOUivTCASW3 (ed .) : I~iedical Fhysiolo~;y (Vol, 1), Z~e C . V . I"Iosby Company, St . Louis (1g68), . D . Z". :GiPSUId and if . LZrFa:AIJ, üetabolism ~ 15y-202 (1974), D . __ . GiaAIIï+r~:i, ~' L . THUl:rSOid and G . i"I . TOIÜ~I ;`iS, J . Biol . Chem . 2~
30. 31 . 32.
(1971) .
1067-1070 (1974) .
1472-1478 (1970) . :.' . J . I? . i:AU':A, J .
Comp .
Ideurol .
104
247-272 (1956),
727-732 12
Life Sciences, Vol . 17, pp . 1607-1616 Printed in U .S .A .
'f l ~ ~FLCTS OF P(.,`5TC7'EP~ATIV :I ^lIt'JD II~JTERVALS AIiU DIURNAL VARIATIG I ON LIVr:R fLYC()~ï~:Iv LFVFhS IPJ nATS :"JI17i Iill'Pï,CAJiPAL 1~ESIOEJS Cyrilla fI . lJi.deman, John Carroll University, Cleveland, Ohio
4411
Helen ;
44118
Thomas S, Brown, DePaul University, Chicago, Illinois
60614
(Received in final form October 27, 1975) Summary Liver glycogen levels were measured in rats with hippocampal lesions and in control animals . Liver glycogen levels were determined each week for the first ten postoperative weeks and eight months postoperatively . It was found that after one week, control animals and animals with hippocampal lesions were not significantly different in liver glycogen levels . By the end of the second week the group with hippocampal lesions was significantly higher than the control animals . This variation pattern continued during the third week . By the end of the third week the animals with hippocampal ablations had reached their highest level, which remained unchanged throughout the rest of the series . No significant changes in liver glycogen levels were obtained in the control animals . Liver glycogen levels were then measured in normal rats and rats with hippocampal lesions maintained on a diurnal rhythm of twelve light hours followed by twelve dark hours . One month postoperatively, rats with hippocampal lesions had a significantly higher liver glycogen level at all time periods as compared with normal animals . Both groups of rats showed the diurnal pattern of higher levels of liver glycogen in the beginning of the light phase and lower levels of liver glycogen in the beginning of the dark phase . The observed variations may be explained in terms of alterations in known homeostatic mechanisms controlling liver glycogen levels . Currently there is an interest in the study of the interrelationship between the hippocampas and the hypothalamic-pituitary-adrenal system . The inhibitôry influence of the hippocampas upon the pituitary-adrenocortical system is well documented . In 1961 Fendler, Karmos and Telegdy (1) reported that extensive lesions of the hippocampas of the cat caused a three-fold increase in secretion of glucocorticoids . Knigge (2~ and Knigge and Hays (3) reported significant increases in basal plasma corticoid levels following bilateral electrolytic hippocampal lesions in rats . Endroczi, Lissak, Bohus and Kovacs (4) and Moberg, Scapagnini, DeGroot and Ganong (5) have domonstrated an inhibitory influence upon the pituitaryadrenocortical system with stimulation of the hippocampas . Mc~wen, ldeiss and Schwartz (6, 7) have shown that radioactive corticosterone injected into rats is taken up by all parts of the brain, but there is a tendency 1607
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Liver Glycogen and Hippocampaj Lesions
Vol . 17, No . 10
for the hippocampus and septum to concentrate and retain the labeled corticosterone . The second study of these authors (7) showed that nuclear retention of the labeled hormone was found to be highest in the hippocampus . In 1972 Gerlach and P"IcEwen (8) showed that when tritiated corticosterone is injected subcutaneously into adrenalectomized male rats one hour before killing, intense labeling of the hippocampus is produced . Glucocorticoids have been shown to have various effects upon protein, carbohydrate and lipid metabolism . Notable among these effects is an increase in enzyme production, especially those enzymes responsible for gluconeogenesis (9, 10, 11, 12) . Weber, Banerjee and Bronstein (13) have shown that cortisone administration increases selectively certain liver enzymes involved in gluconeogenesis, whereas other enzyme systems are not affected, Gleber, Singhal and Stamm (14) have reported that administration of cortisone to rats causes marked de novo synthesis of glucose-6phosphata,se, fructose-l,6-diphosphatase, aldolase and lactic dehydrogenase, all of which are key enzymes in the process of gluconeogenesis . Another important effect of the glucocorticoids is to direct amino acids from tissues to the liver for deamination (15) . In addition, the glucocorticoids have an inhibitory effect on lipogenesis which appears to be of secondary origin and is possibly related to the gluconeogenesis and marked reversal of glycolytic reactions which supply substrates for fat synthesis (15, 16) . The increase in deaminated amino acid residues as well as oxidation fragments (15) entering the glycolytic pathway and Krebs cycle, as well as increases in enzymes concerned with gluconeogenesis, all directly or indirectly due to increased levels of glucocorticoids, would cause a shift in chemical equilibrium resulting in gluconeogenesis . Sie and Fishman (17) have reported that glucocorticoids stimulate glycogen synthetase activity, an enzyme responsible for glycogen synthesis . Thus increased gluconeogenesis and glycogen synthetase activity would result in increased liver glycogen levels with increased circulating glucocorticoids . I'D should be mentioned here that increased levels of glucose-6phosphatase seen with increased levels of circulating glucocorticoids (14) could result in increased blood glucose levels . Murphy, Wideman and Brown (18) did not find an increase in blood glucose levels in animals with hippocampal lesions . In the same study, however, these authors showed that rats with hippocampal lesions had higher liver glycogen concentrations than normal and cortical control animals . The effect of diurnal variation upon liver glycogen levels in rats with hippocampal lesions has not yet been determined . In 1959 Halberg, Peterson and Silber (19) observed a 24-hour rhythm (diurnal variation on a 12 hr . light - 12 hr, dark schedule) of glucocorticoids in mouse serum . The highest level was observed in the eleventh hour of the light period . Similarly, Zimmermann and Critchlow (20) reported that in normal rats there is a diurnal fluctuation in plasma levels of corticosterone with the highest levels being reached in the late afternoon . Their lighting schedule consisted of 14 hrs . of light (from 4c00 a .m . to 6s00 p .m .) followed by 10 hrs, of darkness . In 1966 Slusher (21) implanted cortisol in the ventral hippocampus with a resultant loss in diurnal variation of plasma corticosteroids . Recent data concerning the effects of sectioning the fornix, the major efferent pa~ttsway from the hippocampus, and its influence on plasma corticosterone levels are
Vol . 17, No . 10
Liver Glycogen and Hippocampal Lesions
Whereas Moberg, Scapagnini, DeGroot and Ganong (5) found contradictory . that sectioning of the fornix abolished the diurnal variation in plasma corticosterone levels, Wilson and Critchlow (22) reported that fornix transections were compatible with normal steroid patterns in individual rats . From some of the above studies (1, 2, 3, 4) it has been domonstrated that there are alternations in glucocorticoid levels when the hi pocampus is removed or stimulated . In addition, sectioning the fornix (5~ may These manipulations bring about alterations in glucocorticoid levels . should then have an effect upon both the level and diurnal variation of liver glycogen levels . The present study, therefore, has two purposes : 1) to examine the time course of the increase in liver glycogen levels following ablation of the hippocampus, and 2) to study the effect of 3iurnal variation upon liver glycogen levels in rats with hippocampul lesions. Experiment 1 -_Method Animals The animals used were 114 male Sprague-Dawley rats weighing between 245 and 280 grams at the time of operation. The animals were weigJzed and randomly divided into three groups o£ 38 animals each . The first group received bilateral hippocampul lesions (H) . The second group served as cortical controls (C) and the third group served as normal controls (N) . Bilateral lesions o£ the hippocampus were made as completely as possible by aspirating both medially and ventrally. The overlying cortex was removed on both sides . In cortical control animals only the cortex overlying the hippocampus was removed on both sides by aspiration . Histological Procedure The animals were sacrificed between 8 :00 and 9130 a.m . by decapitation . The brains were immediately removed and placed in lOJ formalin . The brains were embedded in paraffin and sectioned at 15 xt . Every tenth section through the lesion was mounted and stained according to the method o£ Klüver and Barrera (23) " Hippocampal and cortical control lesions were similar to those reported by i~furphy and Brown (24), Extent of damage to the hippocampus ranged from about 70l to 90~ of the structure with most of the ventral tip usually being spared . Experimental Proçedure Animals had ad-lib . access to food and water throughout the experiment and were placed in alternating periods of light and darkness each The experi day. The lights went on at 8:00 a .m . and off at 8s00 p.m . ment was run each week £or the first ten postoperative weeks and eight months postoperatively . For the first five weeks of the experiment four animals from each group were sacrificed at the specified time . For the second five weeks of the experiment as well as for the eight month time period, three animals from each group were sacrificed at the specified time . After decapitation the animals were opened on the ventral side and the liver was removed and placed in a petri dish on ice. The entire liver was weighed and two separate 100 mg . samples were removed from the peripheral third o£ the liver. The liver samples were ground with 10 ml . of 5J trichloroacetic acid in a homogenizer. The homogenate was placed in a The centrifuged homogenate was anatest tube and centrifuged for 5 mir.. lyzed for glycogen according to the iodine method (25) .
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Experiment 1 - Results and Discussion The results of experiment 1 are summarized in Figure 1 . After one week the three groups did not differ significantly in liver glycogen
F
M 7 . W
0 V
F 6
O---O ~"
Normal animal: Hippocompal lodonod animals Q-d Cortical animals
TIME
FIG . 1 Relationship between glycogen concentration and time following lesions . levels . By the end of the second week animals with hippocampal lesions were significantly higher than the control groups . Dy the end of the third week animals with hippocampal ablations had reached their highest level of liver glycogen, This high level remained unchanged throughout the rest of the experiment . Pio significant differences in liver glycogen levels were observed in the control animals . It can be noted that the liver glycogen levels obtained eight months posto eratively replicate the earlier findings of tiurphy, Wideman and Brown (18~ that animals with hippocampal lesions have significantly higher liver glycogen levels than control animals at this time period . Table 1 gives the glycogen concentration in mg . of glycogen per g . of liver for each group at the various time intervals . The numbers have been derived from a standard curve for glycogen concentration . The Mannhihitney U Test showed that animals with bilateral hippocampal lesions ha.d significantly higher levels of liver glycogen than control animals from the second week on . For the second through fifth weeks : H vs . C, U = 0, p = .014 and H vs . N, U = 0, p = .014 . For the sixth throw the tenth weeks and for eight months : H vs . C, U = 0, p = .Oj and Ii vs . N, U = 0, p = .Oj . The animals with hippocampal lesions usually had over twice the amount of liver glycogen as did the other animals from three weeks, until the end of the experiment at eight months . The fact that it took three weeks for this phenomenon to develop indicates that there are biochemical changes occurring in the animals with hippocampal ablations during this time period . From these findings it would appear that such changes should be kept in mind when conducting behavioral studies involving animals with
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Liver Glycogen and Hippocampal Lesions
161 1
hippocampal lesions . In other words, it is suggested that behavioral studies be conducted after these biochemical changes have stabilized . TABLE 1 Summary of ßesults of Liver Glycogen and Postoperative Time Intervals Postop . Group
mg Glycogen per g of Liver
Postop .
Group
mg Glycogen per g of Liver
1 wk .
H G N
322 .5 287 .5 275 .0
7 wks .
II G N
535 " 0 z55 " 0 270 .0
2 wks .
H C N
500 .0 282 .5 250 .0
8 wks .
H G N
570 .0 255.0 275 .0
3 wks .
x C N
650 .0 282 .5 255 .0
9 wks .
x G N
610 .0 287 .5 270 .0
4 wks .
H C N
610 .0 265 .0 275 .o
10 wks .
H C N
510 .0 260 .0 292 .5
5 wks .
H G N
625 .0 285 .5 z5o,o
8 mos .
H c N
610 .0 237 .5 z8z .5
6 wks .
H C N
610 .0 z3o .o 265 .0
Many factors affect the level of liver glycogen in an animal . One factor is food intake of such animals . Boitano, Lubas, Auer and Fernald (26) have shown that rats with hippocampal lesions do not increase food intake . Therefore, the increase in liver glycogen is not brought about by an increase in food intake . A second factor is diurnal variation . Basically, this involves the light-dark cycle . This factor will be considered in experiment 2 . In many cases the two factors overlap in the ra.t . IJith the onset of the dark period the nocturnal rat eats considerably more than in the light cycle . In the present experiment the above two factors were held constant . The animals were fed ad-lib . and were maintained on a 12 hr . light - 12 hr, dark cycle . A third factor is the level of glucocorticoids . Previous studies such as those of Fendler, Karmos and Telegdy (1), Knigge (2) and ICnigge and Hays (3) have shown that the level of glucocorticoids increases in hippocampally-damaged animals . Although Coover, Goldman and Levine (27) did not find an increase of glucocorticoids in animals with hippocampal lesions, their measurement of glucocorticoids followed behavioral experiments which involved water deprivation . Sakellaris and Vernikos-Danellis (28) suggest caution in characterizing animals as normal or adapted in experiments in which some type of stressful regimen such as water deprivation is employed . In the present experiment no stress was introduced into the experimental design . The glucocorticoids have an influence on gluconeogenesis . '~lith an increased level of glucocorticoids the process of gluconeogenesis is speeded up . In addition to gluconeogenesis, the
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Vol . 17, No . 10
glucocorticoids also affect the ability of the liver to store glycogen With an increase of these two activities of the glucocorticoids, (29) . liver glycogen levels should be elevated . Murphy, ~lideman and Brown (18) have shown that liver glycogen levels are significantly higher in rats with hippocampal ablations eight months postoperatively . The present study indicates that increased liver glycogen storage gradually develops over a three week time period in rats with hippocampal lesions . Furthermore, this phenomenon appears to be long lasting rather than temporary. Experiment 2 - Method Animals The animals used were 48 male Sprague-Dawley rats weighing between The animals were weighed and randomly divided into two groups of 24 animals each . The first group received bilateral hippocampal lesions and the second group served as normal controls . A cortical control group was not utilized since previous studies (1£3, experiment 1 of the present study showed that normal and cortical control animals did not differ significantly . Bilateral lesions of the hippocampas were made as completely as possible by aspirating both medially and ventrally . The overlying cortex was removed on both sides.
215 and 245 grams at the onset of the experiment .
Histological and Experimental Procedures The basic histological and experimental procedures were the same as those described in experiment 1 with the following exceptions, The experiment was run one month postoperatively, Three animals from each group were sacrificed at 9 a,m noon, 3, 6, 9 p,m ., midnight, 3 and 6 a,m . Experiment 2 - Results and Discussion A comparison of operated animals and normal animals with respect to liver glycogen levels at various intervals of time is shown in Figure 2, At 9 a .m . the highest liver glycogen level was reached . By 9 p,m. the greatest amount of glycogen had been mobilized from the liver. Although both normal animals and animals with hippocampal lesions maintained diurnal variation in liver glycogen levels, it is apparent from Figure 2 that the latter group possessed markedly higher liver glycogen levels . Within each time interval there were no overlaps of individual animals in the operated group with animals in the normal group. The t4ann-Whitney U Test for each time interval showed that the differences were significant (U = 0, p = ,05) . It is also interesting to note that the general pattern of the two graphs could be readily superimposed upon each other without noticeable variations . Thus both animal groups appear to possess a regulating system which gradually increases or decreases the liver glycogen level . The actual mg, of glycogen per ml . of test solution can be derived from the standard curve of glycogen shown in Figure 3 . On the standard curve only the peak and trough points have been plotted . At 9 a.m . normal animals maintained a glycogen level of 1 .46 mg~ml of test solution . Animals with hippocampal lesions showed a significant increase of liver glycogen at the same time period . The animals reached a peak of 2.24 mg~ml of test solution . Conversely, at 9 p.m . normal animals showed a decrease in liver glycogen to 0,2 mg~ml of test solution . Animals with hippocampal lesions also showed a decrease at 9 p .m but still had a
Vol . 17, No . 10
Liver Glycogen and Hippocampal Lesions
55r 50 45 40 N .35 z ° .30
20 15
.la 05
Pt .05
n 9 om
0
12 noon
n 3 pm
o 6 pm
n n 9 12 pm midn,
n 8 am
3 am
9 am
TIME
FIG .
2
A comparison of hippacampal lesioned and normal animals with respect to diurnal variation in liver glycogen levels . STANDARD CURVE FOR GLYCOGEN
60 .50
O Na~ma1 animale " Hipposampal lesioned animals
a .40 i ° .30 20
t
4
~
B
v
1.2
~
L&
~
2.0
mp GLYCOCaENfmITE51 SOLUTION
_
~2 .4
FIG,
Standard evsve of glycogen concentration showing Beak and trough points of hippocampal lesioned and normal animals.
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Vol . 17, No . 10
significantly higher level of liver glycogen than normal animals, namely, 0 .5 mg~ml of test solution . Values for all the time periods taken from the standard curve are given in Table 2 . TA}3LE 2 Summary of Results of Liver Glycogen during the Diurnal Cycle Time
Group
mg Glycogen per g of Liver
Time
Group
mg Glycogen per g of Liver
9 a .m .
H N
560 .0 365 .0
9 P" m.
x ra
125 .0 50 .0
12 noon
H N
455 .0 335 " o
12 midn .
H N
180 .0 120 .0
3 P .m .
H r7
265 .0 182 .0
3 a .m .
H N
210 .0 180 .0
6 p .m,
H N
220 .0
6 a .m .
H
380 .0 2?5 .0
loo.o
rr
The data presented in this experiment clearly show the effect of diurnal variation on liver glycogen levels, which was not considered in the earlier work of IWrphy, ldideman and Brown (18) . These observations lead one to speculate on the role of the glucocorticoids in the phenomenon of diurnal variation and ultimately the important regulatory part played by the hippocampus . Halberg, Peterson and Silber (19) have reported that the glucocorticoids reach their peak level in the eleventh hour of the light period . In the present experiment this time period corresponds to 7 p .m ., - one hour before the lights go off and before the rat enters into its activity phase . According to Thompson and Lippman (30), the currently popular model for glucocorticoid action states that there is an altered rate of synthesis of certain species of RNA . itibosomal and transfer RNA synthesis are altered depending on the tissue, and presumably there are quantitative and possibly qualitative alterations in the rate of messenger (mRNA) synthesis . These alterations are reflected in altered rates of synthesis of specific proteins (enzymes) . It appears to be established that in most carefully studied examples of enzyme induction by steroids there is a quiet period, or lag, after the hormone reaches the cells, before increased enzyme activity or functional mRNA can be observed (31) " Shortly after the limits o off at 8 .m ., the animal begins to eat, thus increasing the substrate amino acids necessary for gluconeogenic activity . This feeding behavior continues until the lights go on at 8 a .m . Since the level of glucocorticoids is highest one hour before the onset of the dark period, the production of liver gluconeogenic enzymes would slowly increase in quantity . As the digestive process continues in the gut of the rat after eating, the concentration of amino acids increases . ldith increased quantities of amino acids being removed by the liver, and increased quantities of liver gluconeogenic enzymes being produced, the rate of gluconeogenesis would be increased, conceivably reaching its maximum point around 9 a .m ., one hour after the rat has ceased its activity phase . At this point, the glycogen level has peaked . Since the hippocampectomized rat has a diurnal variation pattern similar to ttie normal animal, but in all cases with higher levels of liver glycogen, one could postulate a regulatory role by the hippocampus on
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Liver Glycogen and Hippocampal Lesions
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glucocorticoid secretion . This regulation could possibly be via the hypothalamus since Nauta (32) has reported neuronal connections between the hippocampus and the arcuate nucleus of the hypothalamus in the rat . In summary, it has been shown that diurnal variation affects liver glycogen levels in rats . Although removal of the hippocampus does not alter the diurnal cycle, it appears to have an influence upon the amount of glycogen stored by the liver . Acknowledgment - Supported in part by a Research Grant from the Dept . of Mental Health of the State of Illinois to Thomas S . Brown . Ref erences 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17, 18 . 19 . 20 . 21 . 22 . 23 . 24 . 2$ . 26 .
K, r^EIIDLE3, G . I(ARI"iGS and G . TELr7GDY, Açta Physiol . Açad . Scient . ~ . 20 293-297 (1961) . K . I~Y . IOïIGGE, Proc . Soc . E~ . Biol . hied . 108 18-21 (1961) . h . I'i . IQ3IGGE and I"'i . I-iAYS, Proc . Soc . E~ . Biol . I"Yed . 114 67-68 (1963) " E . ENDRO(:ZI, K . LISSAK, E . BCHiUS and S . KOVAC5, Açta Phvsiol . Açad . Scient . h~ . 16 17-22 (1959) " G . P . MOBFG, Ü. SCAFAGIdI:~I, J . DJGRGOT and 4 . F . GAI~IONG, Idearo endocrinolo~y ~ 11-15 (1971) . B . S . IwiCE:"IfP ;, J . I%i . 'riEISS and L, S . SCHIIAR'T'Z, Lrain i?eso 16 227-241 (1969) . E . S . P4CETIEN, J . I^ . ~~ISS and L . S . SCH ïAÜ1"L, Brain Res . 471-482 (1970) . J . L . GERLACH and B . 5 . I"ICE~IEId, Science ~ 1133-1136 (1972) . E . SHIRAGC, H . A . LARDY, R . C . IdORDLIE and D . 0 . FOSTER, J . Biol . Chem . ~ 3188-31g2 (1963) . G . 17EBER, S . K . SRIVASTAVA and R . L . SINGHAL, J . Biol . Chern . 240 750-756 (1965) " I{ . V . HEIIîiIIdG, id . IfUTrI and I . SEiTBERT, Eiochem . Bio s . Research Communs . ~ 496-501 (1964) . R . J . I4ARTIN, J . A . PATT and R . J . i~BERIiART, Proc . Soc . ~ . Biol . G . I:~~:a;;, G . BAP+c.dJEE and 5 . B . Bï20IiSTEIAI, J . Biol . Chem . 2 ;6 3106-3111 (1961) . G . LdEBra, R . L . SIiJGIIAL and :~' . B . STAMM, Science _l42 390-392 (1963) . ~ . S . >EST, 'r1 . R . TGDD, II . S . I`ïASOIv and J . ^t . VAI B3UGGDri, Textbook of Biochemistry , The I~iacmillan Company, venr York (1966) . R. H . VIILLIAIIS (ed .)s Diabetes , Paul B . Hoeber, Inc ., New York (1960) . H . SIE and ïl . H . FISHI"ïAld, Science 1~ 816-817 (1964) . H . II . i~NRPHY, C . H . 1IDr~4AId and T . S . BRO+:I ;, Phvsiol . Eehav . £; 1171-1174 (1972) . F . i-iALBEPG, i? . E . PETERSGl"1 and R . II . SILi;r.Ft, Endocrinolo~.y ä+ z22-230 (1959) . E . ZTi"uri~?AI1I1 and V . CRiTCIILUIT, _Am . _J . Ph siol . _216 148-155 (1969) . I"I . A . SLUSïIER, ~ . Brain Res . 1 184-194 19 ) . 'i~I . ~dIISOTd and V . CRITCHLO'r1, iveuroendocrinology ~ 29-40 (1974) . H . IIZUVER and E . BARFRA, J . Neuropath . ~ . Neurol . 12 400-403 (1953) " H . i'I . i"IURPHY and T . S . BFtOV1id, J . Comp . Phvsiol . Psychol . Z 404-415 (1970) " J . VAN DER VIES, Biochem , J . ~ 410-416 (1954) . J . J . BOTTANO, J . F . LUBAR, J . AUER and M . S . FERNALD, Physiol . Behau . _3 901-906 (1968) .
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Liver Glycogen and Hippocampal Lesions
27,
G . D. CUUV;JR, L . GULDi:AiQ and S . LEVII~~, i'h:~siol . Behav . ~
28 .
P . C . SAi~LL.~IS and J . V~~idIKUS-DAI~c LLIS, Physiol, i3ehav ,
29 .
V . D, i~IOUivTCASW3 (ed .) : I~iedical Fhysiolo~;y (Vol, 1), Z~e C . V . I"Iosby Company, St . Louis (1g68), . D . Z". :GiPSUId and if . LZrFa:AIJ, üetabolism ~ 15y-202 (1974), D . __ . GiaAIIï+r~:i, ~' L . THUl:rSOid and G . i"I . TOIÜ~I ;`iS, J . Biol . Chem . 2~
30. 31 . 32.
(1971) .
1067-1070 (1974) .
1472-1478 (1970) . :.' . J . I? . i:AU':A, J .
Comp .
Ideurol .
104
247-272 (1956),
727-732 12