RESPIRATION AND OXIDATIVE PHOSPHORYLATION OF THE MITOCHONDRIA OF THE BRAIN OF RATS WITH VARIOUS TYPES OF BEHAVIOR
L. M. Livanova, K. Yu. Sarkisova, L. D. Luk'yanova, and I. A. Koiomeitseva
UDC 612.821.6+612.833.2+612.8.015
A higher level of activity of the succinate oxidase oxidative pathway was found in rats with the active type of behavior (with a high level of motoric activity and a low level of depressiveness) as compared with rats with the passive type of behavior (with a low level of motoric activity combined with low high and medium levels of depressiveness).
The elucidation of the mechanisms underlying the individual-typological features of behavior is one of the vital and complex problems of the physiology of the higher nervous activity of animals. By now data have been obtained which indicate an association of some forms of behavioral reaction with the metabolism of biogenic amines [1, 8, 14-17]. At the same time the energy metabolism of the brain of animals with various types of behavior remains almost unstudied. It has been possible in a few investigations merely to establish a correlation between the activity of some redox enzymes in rabbits and their typological features [5], and to identify the functional differences in the mitochondria of the liver of rats distinguished by the levels of motoric activity and emotionality [6]. In this context the goal of the present study was the investigation of the respiration and oxidative phosphorylation in the mitochondria of the brain of rats with various types of behavior in "open field" and "forced swimming" tests. The hypothesis of the possibility of identifying individual differences in these processes from the point of view of the predominance of reactions of the active or the passive types, assessed by means of these tests, was based on the fact that one of us [12] had previously found two types of shifts in the oxidative metabolism of the brain (in relation to the processes of the transportation and consumption of oxygen by nerve tissue) which correspond to the active (exploratory and active defensive reactions) and passive (passive-defensive freezing reactions) types of behavior. METHODS The experiments were carried out on 33 mongrel male rats weighing 250-300 g. The individual characteristics of the animals' behavior were determined by means of two "open field" and "forced swimming" tests [7, 15]. In order to assess the overall motoric activity the rats were placed for 10 min in the "open field" (an arena with a diameter of 120 cm), and the number of squares crossed, rearings on the hind legs (stands), exits to the center, washings, and defecations was recorded. In order to assess the tendency to manifest a depression-like state the rats were placed for 10 min in a basin with water (diameter of the base 32 cm; height 50 cm; water temperature 20~ and the time during which the rats were in an immobile state (passive swimming). The rats were subdivided appropriately into high- (HA), medium- (MA), and low- (LA) active and into high- (HD), medium- (MD), and low- (LD) depressive animals on the basis of the indices of horizontal motoric activity and passive swimming time. In order to distribute the rats among the groups the average values of the recorded indices were calculated, and, taking the mean square deviation into account, the range of values was determined within the limits of which the motoric activity (or the markedness of the depression-like features in the behavior) was considered medium. Rats in which the motoric activity and passive swimming time values lay beyond the upper and lower bounds of this range were assigned respectively to the high-active (or high-depressive) or the low-active (or low-depressive) groups. The different combinations of the three gradations (low, medium, high) and the two features (general motoric activity level and depressiveness level) made it possible to distinguish in this population, consisting of 33 rats, eight groups of animals (the HA, HD group was lacking). Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, Moscow. Institute of Pharmacology, Russian Academy of Medical Sciences, Moscow. Translated from Zhurnal Vysshei Nervnoi Deyatel'nosti imeni I. P. Pavlova, Vol. 41, No. 5, pp. 973-981, September-October, 1991. Original article submitted November 2, 1990; revision submitted January 9, 1991. 0097-0549/92/2206-0519512.50 9
Plenum Publishing Corporation
519
o
* 74,34• 72,8• 82,t2+3,49
32,54• 1,84 30,t6• 27,79• 27,74-4-3,72 3t,07• t,99 37,39•
HA, MD
LA, MD
LA, LDI
MA, LD
MA, HD
MA, MD
Note: *, p < 0.05; **, p < 0.01; w tendency.
8t,03•
82,28•
8t,3t•
* 7t,9t•
35,03•
LA, HI)
v~
* 87,02•
v~
24,9t•
23,77•
18,97•
21,77•
23,81•
26,86•
22,25•
29,94•
V4
VDNP
85,t4•
87,4~+4,90
87,42•
78,01•
w83,29•
89,54•
~* 77,t7•
:* 93,77•
Rate of respiration
35,62•
Number of animals
HA, LD
Groups of rats
4,tt•
6,58•
5,30•
5,20•
8,96•
8,23•
5,27•
6,75•
VDNp-V3
2,27•
2,72•
3,09•
2,67•
2,58•
* 2,51+0,09
* 2,07•
2,47•
Lardy
3,29•
3,60•
4,53•
3,57+0,22
3,28•
3,03•
3,27•
3,00•
Chance
Respiratory control
0,0i6•
0,0t4•
0,0i9•
0,017•
0,0t5+0,00t3
0,0t7•
0,015•
** 0,0ti•
**
**
Rate of phos-.: phorylation, ADP/T
1,87•
1,79•
2,04•
1,98•
t,79•
2,00•
t,95•
2,05• I
" Coefficient o phosphorylation, ADP/O
TABLE 1. Respiration and Oxidative Phosphorylation of Mitochondria of the Brain of Rats with Various Types of Behavior. Oxidation Substrate Succinate (5 mM)
bo
7
3
4
3
3
5
3
LA, HI)
HA, MD
LA, MD
LA, LD
MA, LD
MA, HD
MA, MD
1,86•
0,017• 3,39• 2,74•
1
2,t9• 93,99•
27,'19• t,45
1t3,89•
91,8•
33,88•
t,88• 0,0t8• 2,65•
4,84•
t0t,09•
25,85•
"t20,58•
95,43•
36,84•
3,76•
0,017 +0,0003 t,85+0,064 3,36• 2,59+0,05
4,47•
94,6i•
27,69•
i10,45•
90,t4•
34,88• 1,38
1,90• 0,(116• 3,47• 4,6t+t,57 * 2,29•
90,45•
25,t3+2,84
85A7•
37,t9•
5 1,83• 0,016• 3,58• 2,74•
t13,it•
93,72+-3,46 4,44• i,20
25,87•
* 120,08•
89,28•
32,62•
t,84• O,Ot6•
3,4t•
4,02+2,021 * 2,75•
91,57•
25,92•
t07,45•
87,55•
3t,88•
1,95• 0,0t7+0,0013 3,27~:0,10 2,57•
6,49•
91,89•
* 26,d1•
* 1t9,64•
85,4t•
34,02•
t,92•
0,018•
2,71•
3,11•
94,79•
Chance
Rate of I~ Coefficient of phosphory-] phosphorylalation, l tion, ADP/O ADP/T I 2,90•
Lardy
Respiratory control VDNp-Va
VDNP
* 32,32•
V4
* 105,02•
VaSA + glu/ VaSA , %
9i,66•
V:~
34,42•
v~
Rate of respiration
Note: SA, 5 mM. glu., 2 mM; *, p < 0.05.
5
HA, LD
Groups ~ ~" of rats ~ o-~
TABLE 2. Respiration and Oxidative Phosphorylation of Mitochondria of the Brain of Rats with Various Types of Behavior. Oxidation Substrate Succinate (succinic acid - SA) with Glutamate (glu)
tO tO
5
7
3
4
3
3
5
3
HA, LD
LA, HD
HA, MD
LA, MD
LA, LD
MA, LD
MA, HD
MA, MD
of rats i ~ ~
5,34• 4,77-+0,3t 5,20• 4,89-+0,54 4,48-+0,65 5,39•
3,5i+0,25 3,35-+0,08 3,27• 3,i5• 3,i0• 3,77•
1,49-+0,75 3,16• i,98• 2,38-+t,t3
32,75• 28,86tt 39 3Lt4• 30,96•
5,98• 4,75• 5,63• 6,09•
7,66+0,56 25,69• 29,i7•
4,43• 4,48•
3,53• 3,29•
3,81• 238• 4,t5• 2,64-+0,85
32,84• 32,i3• 33,i4• 3i,93•
6,75• i,07 5,83• 6,69• 7,28•
29,03• 29,94• 28,99• 30,85•
9,39•
8,i1+i,3~
8,34•
9,83•
9,12-+0,71 28,58• t,89
8,90•
31,26•
8,95•
Chance
Lardy
v4
v~
v,.
VDNp-V3
Respiratnry control
VDNP
9Rate of respiration
2,85• 2,89• 2,87+0,08
2,89• 2,84•
0,0083-+0,0007 0,0083• 0,0083.+0,0008 0,0086-+0,0009 0,0083+0,0005 0,0088•
32,78• 32,86• 4L44-+7,70 40,97• 37,92• 37,93-+2,71
2,87•
2,9t• 0,0074+0,0005
3i,54•
2,92• 0,009i-+0,0007
33,58•
acid, %
Endogenous Rate of iCoefficient pool of ,phosphoryla- of phosphorysuccinic tion, ADP/T lation, ADP/O
TABLE 3. Respiration and Oxidative Phosphorylation of Mitochondria of the Brain of Rats with Various Types of Behavior. Oxidation Substrate Glutamate (4 mM) with Malate (2 raM)
After the individual characteristics of the behavior of the rats were determined they were maintained in standard vivarium conditions for 10 days. The rats were then decapitated with careful observation of conditions excluding their being stressed prior to sacrifice. The brain mitochondria were obtained by the technique of differential centrifugation, taking into account modifications guaranteeing the preservation of the native condition of the isolated organelles and the identification of their physiological differences in vitro conditions [2, 3]. The isolation medium for mitochondria contained (in mM): sucrose, 280; tris-HC1, 10 (pH 7.4); EDTA (ethylenediamine acetate), 1; BSA (bovine serum albumin), 0.2%. The resuspension medium had the same composition with the exception of EDTA. The rate of oxygen absorption by the mitochondria was recorded polarigraphically with the aid of an open rotating platinum electrode at a temperature of 26~ [11]. The incubation medium contained (in mM): sucrose, 280; KCI, 10; tris-HC1, 10 (pH 7.4); EDTA, 1; KH2PO4, 3; BSA, 0.2%. The following were used as the oxidation substrates: succinate (5 raM), a mixture of succinate and glutamate (2 raM), and a mixture of glutamate (4 mM) and malate (2 mM). The mitochondria (2-3 mg of protein) were introduced into the medium containing the subsltrate, and the respiratory rates V2 (ng at. O/(min 9mg protein)), V 3 (after the introduction of 200 glVl ADP, in the metabolically active state), V 4 (at the end of phosphorylation), and VDNP (in the presence of the oxidative phosphorylation uncoupler 2,4-DNP) were recorded. The magnitude of the endogenous succinate pool was assessed on the basis on the degree of inhibition of the rate of uncoupled respiration by the succinate dehydrogenase inhibitor, malonate (2 mM), during the oxidation of the glutamate-malate mixture. The phosphorylation index, ADP/O, which characterizes the degree of coupling of the processes of respiration and phosphorylation, the ADP phosphorylation rate (M/(min. mg protein) and values of the Lardy respiratory controls (RC L, the V3/V 2 respiratory rates ratio) and the Chance respiratory controls (RC C, the V3/V 4 respiratory rates ratio) [13], and the VDNp-V 3 respiratory rates difference were calculated. The protein of the mitochondria was determined using the biuret reagent. All the data were analyzed statistically by the Student method. INVESTIGATION RESULTS When succinate was used as the oxidation substrate (Table 1), significant differences in respiration and oxidative phosphorylation of the brain mitochondria could be identified between the rats with the active type of behavior (with high motoric activity and a low depressivenesss level, the HA, LD group) and rats with the passive type of behavior (with tow motoric activity combined with high and medium depressiveness levels, the LA, HD and LA, MD groups). The brain mitochondria of rats with the active type of behavior had higher V3and VDNP respiratory rates and ADP phosphorylation rate. Differences were not found in the ADP/O value between the groups of animals with the active and passive types of behavior. VDNp--V3.respiratory rates differences, which characterize the "reserve" of the respiratory activity of the mitochondria were not distinguished statistically significantly in preparations of mitochondria isolated from the brain of rats with the active and passive types of behavior. Differences were also not detected between these groups of rats with respect to the V 2 and V 4 respiratory rates and with respect to the RC C value. The RC L was lower in rats of the LA, HD group as compared with the rats of the active type with medium depressiveness. When the succinate-glutamate mixture was used as the oxidation substrates (Table 2), the differences which were found with the oxidation of succinate alone were not identified in the respiration and oxidative phosphorylation of the mitochondria of the brain of rats with the active and passive types of behavior. It was established that the V 4 respiratory rate of the mitochondria of rats with the active type of behavior was insignificantly higher than the analogous index of the mitochondria of rats with the passive type of behavior. At the same time the degree of increase in the V 3 respiratory rate when glutamate was added to succinate was significantly higher in the mitochondria of rats with low and medium motoric activity which is combined with high and low levels of depressiveness, as compared with rats which are characterized by high motoric activity and a low level of depressiveness. When NAD-dependant substrates (glutamate and malate) were oxidized, significant differences were not identified in respiration and oxidative phosphorylation of the mitochondria of the brain of rats with various types of behavior (Table 3). DISCUSSION OF RESULTS It follows from the data presented that differences in respiration and oxidative phosphorylation of the mitochondria of the brain of animals with various types of behavior were manifested most distinctly when succinate is oxidized. The data obtained a higher level of succinate oxidase and ATP synthetase activity and degree of energization of the mitochondria of the brain of rats with the active type of behavior as compared with rats with the passive type of behavior. A relatively greater stimulation of the V 3 respiratory rate was identified in the latter when a NAD-dependant substrate, glutamate, was added to
523
the succinate. It is conventional to explain the activating influence of glutamate on the oxidation of succinate by the removal of oxaloacetic acid, which exerts an inhibiting influence on succinate dehydrogenase, and in certain cases accumulates in the mitochondria during the oxidation of succinate [2, 4]. It can also be hypothesized that monopolization of the respiratory chain by succinate does not take place in the presence of relatively lower activity of succinate oxidase oxidation pathway which is characteristic for animals with the passive type of behavior, and that the conditions are thereby created for the simultaneous oxidation of NAD-dependant substrates and succinate. On the other hand, in the mitochondria of the brain of rats with the active type of behavior, which are distinguished by a relatively greater succinate oxidase activity and which exhibit a high respiratory rate, the effect of the monopolization of the respiratory chain by succinate may take place. In this case, as can be seen from our data, the addition of a NAD-dependant substrate does not exert an influence on the respiratory rate of mitochondria which oxidize succinate. Our results coincide in the main with data obtained in an investigation of mitochondria of the liver of rats with various types of behavior in the open field [6]. In low-emotional rats with a high level of motoric activity, the mitochondria of the liver exhibit a higher V3xcspiratory rate during the oxidation of succinate, the phosphorylation time was shorter, and the Lardy and Chance respiratory controls were somewhat higher than in the mitochondria of highly-emotional rats with low level of motoric activity. Significant differences were identified only in the phosphorylation time when glutamate was oxidized. The shift to the preferential oxidation of succinate had been observed previously under conditions of catecholamine excitation, in the presence of stress [3, 4]. It should be noted that the higher level of activity of the succinate oxidase oxidation pathway in our experiments in the mitochondria of the brain of rats with the active type of behavior was identified in the tranquil state of the animals prior to sacrifice, and in the absence of any functional loads. It may be hypothesized that the varying degree of the activity of the succinate oxidase oxidation pathway in the mitochondria of animals with the active and passive types of behavior is governed by varying neurohumoral regulation of succinate dehydrogenase. According to published data, its activators are epinephrine, norepinephrine, and dopamine [4]. Antagonists of this group of hormones, acetylcholine and serotonin, affect succinate dehydrogenase oppositely [2-4]. It has been demonstrated that the activity of the norepinephrinergic and dopaminergic systems of the brain correlates positively with the active type of behavior in the "open field" and "forced swimming" tests and negatively with the passive type of behavior [1, 8, 14, 15], while the activity of the cholinergic and serotoninergic systems of the brain correlates positively with the passive type of behavior and negatively with the active type of behavior ]1, 8,16, 17]. It has also been demonstrated that chronic deprivation of the activity of the catecholaminergic systems induced by the administration of 6-hydroxydopamine to newborn rats leads to a decrease in the activity of succinate dehydrogenase in the brain structures of adult rats which is accompanied by a suppression of motoric activity in the open field [10]. Thus, a correlation has been identified in our experiments between the level of the activity of the succinate oxidase oxidation pathway in the mitochondria of the brain of rats and their individual-typological features of behavior. It should be noted that the varied activity of the metabolic streams at the substrate end of the respiratory chain had been previously established for the brain of animals with nonidentical resistance to oxygen deficit [9]. The data obtained make it possible to conclude that the features of energy metabolism we found in the mitochondria of the brain, and in particular the specific characteristics of the utilization of the oxidation substrates of the respiratory chain, may exert an influence on the predominance of the active or passive type of behavior of the animals. CONCLUSIONS 1. Significant differences in the functional state of the mitochondria of the brain have been found in an investigation of eight groups of rats with various combinations of levels of motoric activity and depressiveness, between groups of rats with the active type of behavior (with a high motoric activity level and low depressiveness level) and with the passive type of behavior (with a low motoric activity level, combined with high and medium depressiveness levels). 2. A higher level of the activity of the succinate oxidase oxidation pathway was found in the mitochondria of the brain of rats with the active type of behavior as compared with the rats characterized by the passive type of behavior. 3. When NAD-dependant substrates (glutamate and malate) were oxidized, significant differences in respiration and oxidative phosphorylation were not detected in the mitochondria of the brain of rats with various types of behavior.
524
LITERATURE CITED .
.
.
4.
.
6.
.
8. 9.
10.
11. 12. 13. 14. 15. 16. 17.
Kh. Yu. Ismailova, G. G. Gasanov, T. R Semenova, et al., "The influence of the local injection of 5,7-DHT and 6H_DAinto the neoc0rtex on learning and investigatory behavior of rats in the open field," Zh. Vyssh. Nerv. Deyat., 39, No. 3, 548-555 (1989). M. N. Kondrashova and E. V. Grigorenko, Defense against Stress at the Level of the Mitochondria: Development of the Oxaloacetic Limitation of Respiration of Mitochondria Under Prolonged Stress and with the Administration of Serotonin [in Russian], ONTI NTsBI, Pushehino (1981). M. N. Kondrashova and E. V. Grigorenko, "The manifestation of stress at the level of the mitochondria, their stimulation by hormones, and the regulation of aerohydro-ions," Zh. Obshch. Biologii, 36, No. 4, 516-526 (1985). M. N. Kondrashova, E. V. Grigorenko, A. M. Babskii, et al.,"The achievement of homeostasis of physiological functions at the level of the mitochondria," in: The Molecular Mechanisms of Cellular Homeostasis [in Russian], Nauka, Novosibirsk (1987), pp. 40--66. M. 1~. Krakovskii, "The activity of junctional redox enzymes in rabbits with various typological characteristics," Zh. Vyssh. Nerv. Deyat., 37, No. 3, 457-461 (1987). M. 1~. Krakovskii, Ts. L. Kamenetskaya, G. Ya. Tremasova, et al., "The characteristics of certain biochemical processes in the liver of rats with various types of behavior in the open field," Zh. Vyssh. Nerv. Deyat., 39, No. 3,506512 (1989). D. A. Kulagin and V. K. Fedorov, "Investigation of emotionality in rats of the Wistar and Krushinskii-Molodkina lines by the 'open field' method," in: The Genetics of Behavior [in Russian], Nauka, Leningrad (1969), pp. 35-42. D. A. Kulagin and V. K. Bolondinskii, "Neurochemical aspects of emotional reactivity and motoric activity of rats in a new situation," Usp. Fiziol. Nauk, 17, No. 1, 92-109 (1986). L. D. Luk'yanova and A. V. Korobkov, "Some physiological and metabolic characteristics of the individual sensitivity of animals to hypoxia in the norm and in adaptation," in: Summaries of the II1 All-Union Symposium "Physiological and Clinical Problems of Adaptation to Hypoxia, Hypodynamia, and Hyperthermia" [in Russian], Moscow (1981), pp. 73-76. E. B. Okon, T. P. Semenova, and M. I. Grishchenko, "The suppression of energy exchange in the cerebral cortex of rats during deprivation of catecholaminergic systems," in: The Metabolic Regulation of the Physiological State [in Russian], Pushchino (1984), p. 67. Manual for the Study of Biological Oxidation by the Polarographic Method, G. M. Frank (ed.) [in Russian], Nauka, Moscow (1973). K. Yu. Sarkisova, "Change in a level of oxygen tension in various structures of the brain in rats in positive and negative emotional states," Zh. Vyssh. Nerv. Deyat., 40, No. 1,351-361 (1990). B. Chance and G. Hollunger, "The interaction of energy and electron transfer reactions in mitochondria," J. Biol. Chem., 230, No. 5, 1534-1543 (1961). D. V. Jeste, and G. P. Smith, "Unilateral mesolimbicocortical dopamine denervation decreases locomotion in the open field and after amphetamine," Pharmacol. Biochem. Behav., 12, No. 3,453-457 (1980). R. D. Porsolt, A. Bertin, N. Blavet, et al., "Immobility induced by forced swimming in rats: effects of agents which modify central catecholamine and serotonin activity," Eur. J. Pharmacol., 57, 201-210 (1979). H. S. Sudac and J. W. Maas, "Behavioral-neurochemical correlation in reactive and nonreactive strains of rats," Science, 146, No. 3642, 418420 (1964). J. H. F. Van Abeelen, "Genotype and the cholinergic control of exploratory behavior in mice," in: The Genetics of Behavior, J. H. F. Van Abeelen (ed.), Amsterdam (1974), pp. 346-374.
525
L. M. Livanova, K. Yu. Sarkisova, L. D. Luk'yanova, and I. A. Koiomeitseva
UDC 612.821.6+612.833.2+612.8.015
A higher level of activity of the succinate oxidase oxidative pathway was found in rats with the active type of behavior (with a high level of motoric activity and a low level of depressiveness) as compared with rats with the passive type of behavior (with a low level of motoric activity combined with low high and medium levels of depressiveness).
The elucidation of the mechanisms underlying the individual-typological features of behavior is one of the vital and complex problems of the physiology of the higher nervous activity of animals. By now data have been obtained which indicate an association of some forms of behavioral reaction with the metabolism of biogenic amines [1, 8, 14-17]. At the same time the energy metabolism of the brain of animals with various types of behavior remains almost unstudied. It has been possible in a few investigations merely to establish a correlation between the activity of some redox enzymes in rabbits and their typological features [5], and to identify the functional differences in the mitochondria of the liver of rats distinguished by the levels of motoric activity and emotionality [6]. In this context the goal of the present study was the investigation of the respiration and oxidative phosphorylation in the mitochondria of the brain of rats with various types of behavior in "open field" and "forced swimming" tests. The hypothesis of the possibility of identifying individual differences in these processes from the point of view of the predominance of reactions of the active or the passive types, assessed by means of these tests, was based on the fact that one of us [12] had previously found two types of shifts in the oxidative metabolism of the brain (in relation to the processes of the transportation and consumption of oxygen by nerve tissue) which correspond to the active (exploratory and active defensive reactions) and passive (passive-defensive freezing reactions) types of behavior. METHODS The experiments were carried out on 33 mongrel male rats weighing 250-300 g. The individual characteristics of the animals' behavior were determined by means of two "open field" and "forced swimming" tests [7, 15]. In order to assess the overall motoric activity the rats were placed for 10 min in the "open field" (an arena with a diameter of 120 cm), and the number of squares crossed, rearings on the hind legs (stands), exits to the center, washings, and defecations was recorded. In order to assess the tendency to manifest a depression-like state the rats were placed for 10 min in a basin with water (diameter of the base 32 cm; height 50 cm; water temperature 20~ and the time during which the rats were in an immobile state (passive swimming). The rats were subdivided appropriately into high- (HA), medium- (MA), and low- (LA) active and into high- (HD), medium- (MD), and low- (LD) depressive animals on the basis of the indices of horizontal motoric activity and passive swimming time. In order to distribute the rats among the groups the average values of the recorded indices were calculated, and, taking the mean square deviation into account, the range of values was determined within the limits of which the motoric activity (or the markedness of the depression-like features in the behavior) was considered medium. Rats in which the motoric activity and passive swimming time values lay beyond the upper and lower bounds of this range were assigned respectively to the high-active (or high-depressive) or the low-active (or low-depressive) groups. The different combinations of the three gradations (low, medium, high) and the two features (general motoric activity level and depressiveness level) made it possible to distinguish in this population, consisting of 33 rats, eight groups of animals (the HA, HD group was lacking). Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, Moscow. Institute of Pharmacology, Russian Academy of Medical Sciences, Moscow. Translated from Zhurnal Vysshei Nervnoi Deyatel'nosti imeni I. P. Pavlova, Vol. 41, No. 5, pp. 973-981, September-October, 1991. Original article submitted November 2, 1990; revision submitted January 9, 1991. 0097-0549/92/2206-0519512.50 9
Plenum Publishing Corporation
519
o
* 74,34• 72,8• 82,t2+3,49
32,54• 1,84 30,t6• 27,79• 27,74-4-3,72 3t,07• t,99 37,39•
HA, MD
LA, MD
LA, LDI
MA, LD
MA, HD
MA, MD
Note: *, p < 0.05; **, p < 0.01; w tendency.
8t,03•
82,28•
8t,3t•
* 7t,9t•
35,03•
LA, HI)
v~
* 87,02•
v~
24,9t•
23,77•
18,97•
21,77•
23,81•
26,86•
22,25•
29,94•
V4
VDNP
85,t4•
87,4~+4,90
87,42•
78,01•
w83,29•
89,54•
~* 77,t7•
:* 93,77•
Rate of respiration
35,62•
Number of animals
HA, LD
Groups of rats
4,tt•
6,58•
5,30•
5,20•
8,96•
8,23•
5,27•
6,75•
VDNp-V3
2,27•
2,72•
3,09•
2,67•
2,58•
* 2,51+0,09
* 2,07•
2,47•
Lardy
3,29•
3,60•
4,53•
3,57+0,22
3,28•
3,03•
3,27•
3,00•
Chance
Respiratory control
0,0i6•
0,0t4•
0,0i9•
0,017•
0,0t5+0,00t3
0,0t7•
0,015•
** 0,0ti•
**
**
Rate of phos-.: phorylation, ADP/T
1,87•
1,79•
2,04•
1,98•
t,79•
2,00•
t,95•
2,05• I
" Coefficient o phosphorylation, ADP/O
TABLE 1. Respiration and Oxidative Phosphorylation of Mitochondria of the Brain of Rats with Various Types of Behavior. Oxidation Substrate Succinate (5 mM)
bo
7
3
4
3
3
5
3
LA, HI)
HA, MD
LA, MD
LA, LD
MA, LD
MA, HD
MA, MD
1,86•
0,017• 3,39• 2,74•
1
2,t9• 93,99•
27,'19• t,45
1t3,89•
91,8•
33,88•
t,88• 0,0t8• 2,65•
4,84•
t0t,09•
25,85•
"t20,58•
95,43•
36,84•
3,76•
0,017 +0,0003 t,85+0,064 3,36• 2,59+0,05
4,47•
94,6i•
27,69•
i10,45•
90,t4•
34,88• 1,38
1,90• 0,(116• 3,47• 4,6t+t,57 * 2,29•
90,45•
25,t3+2,84
85A7•
37,t9•
5 1,83• 0,016• 3,58• 2,74•
t13,it•
93,72+-3,46 4,44• i,20
25,87•
* 120,08•
89,28•
32,62•
t,84• O,Ot6•
3,4t•
4,02+2,021 * 2,75•
91,57•
25,92•
t07,45•
87,55•
3t,88•
1,95• 0,0t7+0,0013 3,27~:0,10 2,57•
6,49•
91,89•
* 26,d1•
* 1t9,64•
85,4t•
34,02•
t,92•
0,018•
2,71•
3,11•
94,79•
Chance
Rate of I~ Coefficient of phosphory-] phosphorylalation, l tion, ADP/O ADP/T I 2,90•
Lardy
Respiratory control VDNp-Va
VDNP
* 32,32•
V4
* 105,02•
VaSA + glu/ VaSA , %
9i,66•
V:~
34,42•
v~
Rate of respiration
Note: SA, 5 mM. glu., 2 mM; *, p < 0.05.
5
HA, LD
Groups ~ ~" of rats ~ o-~
TABLE 2. Respiration and Oxidative Phosphorylation of Mitochondria of the Brain of Rats with Various Types of Behavior. Oxidation Substrate Succinate (succinic acid - SA) with Glutamate (glu)
tO tO
5
7
3
4
3
3
5
3
HA, LD
LA, HD
HA, MD
LA, MD
LA, LD
MA, LD
MA, HD
MA, MD
of rats i ~ ~
5,34• 4,77-+0,3t 5,20• 4,89-+0,54 4,48-+0,65 5,39•
3,5i+0,25 3,35-+0,08 3,27• 3,i5• 3,i0• 3,77•
1,49-+0,75 3,16• i,98• 2,38-+t,t3
32,75• 28,86tt 39 3Lt4• 30,96•
5,98• 4,75• 5,63• 6,09•
7,66+0,56 25,69• 29,i7•
4,43• 4,48•
3,53• 3,29•
3,81• 238• 4,t5• 2,64-+0,85
32,84• 32,i3• 33,i4• 3i,93•
6,75• i,07 5,83• 6,69• 7,28•
29,03• 29,94• 28,99• 30,85•
9,39•
8,i1+i,3~
8,34•
9,83•
9,12-+0,71 28,58• t,89
8,90•
31,26•
8,95•
Chance
Lardy
v4
v~
v,.
VDNp-V3
Respiratnry control
VDNP
9Rate of respiration
2,85• 2,89• 2,87+0,08
2,89• 2,84•
0,0083-+0,0007 0,0083• 0,0083.+0,0008 0,0086-+0,0009 0,0083+0,0005 0,0088•
32,78• 32,86• 4L44-+7,70 40,97• 37,92• 37,93-+2,71
2,87•
2,9t• 0,0074+0,0005
3i,54•
2,92• 0,009i-+0,0007
33,58•
acid, %
Endogenous Rate of iCoefficient pool of ,phosphoryla- of phosphorysuccinic tion, ADP/T lation, ADP/O
TABLE 3. Respiration and Oxidative Phosphorylation of Mitochondria of the Brain of Rats with Various Types of Behavior. Oxidation Substrate Glutamate (4 mM) with Malate (2 raM)
After the individual characteristics of the behavior of the rats were determined they were maintained in standard vivarium conditions for 10 days. The rats were then decapitated with careful observation of conditions excluding their being stressed prior to sacrifice. The brain mitochondria were obtained by the technique of differential centrifugation, taking into account modifications guaranteeing the preservation of the native condition of the isolated organelles and the identification of their physiological differences in vitro conditions [2, 3]. The isolation medium for mitochondria contained (in mM): sucrose, 280; tris-HC1, 10 (pH 7.4); EDTA (ethylenediamine acetate), 1; BSA (bovine serum albumin), 0.2%. The resuspension medium had the same composition with the exception of EDTA. The rate of oxygen absorption by the mitochondria was recorded polarigraphically with the aid of an open rotating platinum electrode at a temperature of 26~ [11]. The incubation medium contained (in mM): sucrose, 280; KCI, 10; tris-HC1, 10 (pH 7.4); EDTA, 1; KH2PO4, 3; BSA, 0.2%. The following were used as the oxidation substrates: succinate (5 raM), a mixture of succinate and glutamate (2 raM), and a mixture of glutamate (4 mM) and malate (2 mM). The mitochondria (2-3 mg of protein) were introduced into the medium containing the subsltrate, and the respiratory rates V2 (ng at. O/(min 9mg protein)), V 3 (after the introduction of 200 glVl ADP, in the metabolically active state), V 4 (at the end of phosphorylation), and VDNP (in the presence of the oxidative phosphorylation uncoupler 2,4-DNP) were recorded. The magnitude of the endogenous succinate pool was assessed on the basis on the degree of inhibition of the rate of uncoupled respiration by the succinate dehydrogenase inhibitor, malonate (2 mM), during the oxidation of the glutamate-malate mixture. The phosphorylation index, ADP/O, which characterizes the degree of coupling of the processes of respiration and phosphorylation, the ADP phosphorylation rate (M/(min. mg protein) and values of the Lardy respiratory controls (RC L, the V3/V 2 respiratory rates ratio) and the Chance respiratory controls (RC C, the V3/V 4 respiratory rates ratio) [13], and the VDNp-V 3 respiratory rates difference were calculated. The protein of the mitochondria was determined using the biuret reagent. All the data were analyzed statistically by the Student method. INVESTIGATION RESULTS When succinate was used as the oxidation substrate (Table 1), significant differences in respiration and oxidative phosphorylation of the brain mitochondria could be identified between the rats with the active type of behavior (with high motoric activity and a low depressivenesss level, the HA, LD group) and rats with the passive type of behavior (with tow motoric activity combined with high and medium depressiveness levels, the LA, HD and LA, MD groups). The brain mitochondria of rats with the active type of behavior had higher V3and VDNP respiratory rates and ADP phosphorylation rate. Differences were not found in the ADP/O value between the groups of animals with the active and passive types of behavior. VDNp--V3.respiratory rates differences, which characterize the "reserve" of the respiratory activity of the mitochondria were not distinguished statistically significantly in preparations of mitochondria isolated from the brain of rats with the active and passive types of behavior. Differences were also not detected between these groups of rats with respect to the V 2 and V 4 respiratory rates and with respect to the RC C value. The RC L was lower in rats of the LA, HD group as compared with the rats of the active type with medium depressiveness. When the succinate-glutamate mixture was used as the oxidation substrates (Table 2), the differences which were found with the oxidation of succinate alone were not identified in the respiration and oxidative phosphorylation of the mitochondria of the brain of rats with the active and passive types of behavior. It was established that the V 4 respiratory rate of the mitochondria of rats with the active type of behavior was insignificantly higher than the analogous index of the mitochondria of rats with the passive type of behavior. At the same time the degree of increase in the V 3 respiratory rate when glutamate was added to succinate was significantly higher in the mitochondria of rats with low and medium motoric activity which is combined with high and low levels of depressiveness, as compared with rats which are characterized by high motoric activity and a low level of depressiveness. When NAD-dependant substrates (glutamate and malate) were oxidized, significant differences were not identified in respiration and oxidative phosphorylation of the mitochondria of the brain of rats with various types of behavior (Table 3). DISCUSSION OF RESULTS It follows from the data presented that differences in respiration and oxidative phosphorylation of the mitochondria of the brain of animals with various types of behavior were manifested most distinctly when succinate is oxidized. The data obtained a higher level of succinate oxidase and ATP synthetase activity and degree of energization of the mitochondria of the brain of rats with the active type of behavior as compared with rats with the passive type of behavior. A relatively greater stimulation of the V 3 respiratory rate was identified in the latter when a NAD-dependant substrate, glutamate, was added to
523
the succinate. It is conventional to explain the activating influence of glutamate on the oxidation of succinate by the removal of oxaloacetic acid, which exerts an inhibiting influence on succinate dehydrogenase, and in certain cases accumulates in the mitochondria during the oxidation of succinate [2, 4]. It can also be hypothesized that monopolization of the respiratory chain by succinate does not take place in the presence of relatively lower activity of succinate oxidase oxidation pathway which is characteristic for animals with the passive type of behavior, and that the conditions are thereby created for the simultaneous oxidation of NAD-dependant substrates and succinate. On the other hand, in the mitochondria of the brain of rats with the active type of behavior, which are distinguished by a relatively greater succinate oxidase activity and which exhibit a high respiratory rate, the effect of the monopolization of the respiratory chain by succinate may take place. In this case, as can be seen from our data, the addition of a NAD-dependant substrate does not exert an influence on the respiratory rate of mitochondria which oxidize succinate. Our results coincide in the main with data obtained in an investigation of mitochondria of the liver of rats with various types of behavior in the open field [6]. In low-emotional rats with a high level of motoric activity, the mitochondria of the liver exhibit a higher V3xcspiratory rate during the oxidation of succinate, the phosphorylation time was shorter, and the Lardy and Chance respiratory controls were somewhat higher than in the mitochondria of highly-emotional rats with low level of motoric activity. Significant differences were identified only in the phosphorylation time when glutamate was oxidized. The shift to the preferential oxidation of succinate had been observed previously under conditions of catecholamine excitation, in the presence of stress [3, 4]. It should be noted that the higher level of activity of the succinate oxidase oxidation pathway in our experiments in the mitochondria of the brain of rats with the active type of behavior was identified in the tranquil state of the animals prior to sacrifice, and in the absence of any functional loads. It may be hypothesized that the varying degree of the activity of the succinate oxidase oxidation pathway in the mitochondria of animals with the active and passive types of behavior is governed by varying neurohumoral regulation of succinate dehydrogenase. According to published data, its activators are epinephrine, norepinephrine, and dopamine [4]. Antagonists of this group of hormones, acetylcholine and serotonin, affect succinate dehydrogenase oppositely [2-4]. It has been demonstrated that the activity of the norepinephrinergic and dopaminergic systems of the brain correlates positively with the active type of behavior in the "open field" and "forced swimming" tests and negatively with the passive type of behavior [1, 8, 14, 15], while the activity of the cholinergic and serotoninergic systems of the brain correlates positively with the passive type of behavior and negatively with the active type of behavior ]1, 8,16, 17]. It has also been demonstrated that chronic deprivation of the activity of the catecholaminergic systems induced by the administration of 6-hydroxydopamine to newborn rats leads to a decrease in the activity of succinate dehydrogenase in the brain structures of adult rats which is accompanied by a suppression of motoric activity in the open field [10]. Thus, a correlation has been identified in our experiments between the level of the activity of the succinate oxidase oxidation pathway in the mitochondria of the brain of rats and their individual-typological features of behavior. It should be noted that the varied activity of the metabolic streams at the substrate end of the respiratory chain had been previously established for the brain of animals with nonidentical resistance to oxygen deficit [9]. The data obtained make it possible to conclude that the features of energy metabolism we found in the mitochondria of the brain, and in particular the specific characteristics of the utilization of the oxidation substrates of the respiratory chain, may exert an influence on the predominance of the active or passive type of behavior of the animals. CONCLUSIONS 1. Significant differences in the functional state of the mitochondria of the brain have been found in an investigation of eight groups of rats with various combinations of levels of motoric activity and depressiveness, between groups of rats with the active type of behavior (with a high motoric activity level and low depressiveness level) and with the passive type of behavior (with a low motoric activity level, combined with high and medium depressiveness levels). 2. A higher level of the activity of the succinate oxidase oxidation pathway was found in the mitochondria of the brain of rats with the active type of behavior as compared with the rats characterized by the passive type of behavior. 3. When NAD-dependant substrates (glutamate and malate) were oxidized, significant differences in respiration and oxidative phosphorylation were not detected in the mitochondria of the brain of rats with various types of behavior.
524
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11. 12. 13. 14. 15. 16. 17.
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