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UDC 612.4.03:612.821
BLOOD CORTICOSTERONE AND HIPPOCAMPAL NORADRAENALINE BY DIFFERENT STRATEGY OF THE RESULT-BASED BEHAVIOR: ASSESSMENT OF BEHAVIOR IN THE CPAR TEST FROM THE PERSPECTIVE OF THE THEORY OF FUNCTIONAL SYSTEMS
1 I.M. Sechenov First Moscow State Medical University, Moscow
2 P.K. Anokhin Research Institute of Normal Physiology, Moscow
D.I. Babaevskaya1, S.A. Kalinin1, N.Yu. Chekmareva12, A.E. Umryukhin12
Functional systems theory proposed by P.K. Anokhin and elaborated by K.V. Sudakov suggests the achievement of the necessary result as the main determinant factor of physiological responses to external and internal stimuli. Based on this concept it may be concluded that passive avoidance test reveals different strategies of reward achievement behavior of animals. Our data show that passive strategy of aversive stimulus avoidance behavior is accompanied by a stable unchangeable profile of hippocampal noradrenalin under immobilization stress exposure and after its termination as well as by a stable unchangeable blood corticosterone level after stress. Active strategy of aversive stimulus non-escaping behavior in this test is accompanied by an increase of hippocampal noradrenalin during immobilization stress exposure and after its termination as well as by an increase of blood corticosterone level after immobilization stress.
Key words: functional systems theory, stress, behavior, hippocampus, noradrenalin, corticosterone, microdialysis, ELISA.
The study of cerebral mechanisms of psychoemo-tional stress and the search for ways to increase individual resistance to the negative consequences of emotional stress is an actual task of modern science [4]. Behavioral activity in the «open field» test reflects the individual characteristics emotionally anxious qualities [14] and may serve as predictor of individual resistance to emotional stress [4]. The specimens active in the test «open field» under similar stress loads are showing signs of stress resistance to the action of individuals, while passive -prone [1]. It is known that the characteristics of behavior, learning and memory, the nature and extent of abuses against the background of stress loads are closely related to brain processes regulating the activity of the hypothalamic-pituitary-adrenal axis and levels of corticosterone [9, 15, 18]. An important role is performed overhypothalamic centers regulating the activity of the paraventricular nucleus of the hypothalamus - the dorsal hippocampus and noradrenergic brain mechanisms [20]. In connection with this, the objective of the study was to examine the behavior of rats with different behavioral activity in the test «conditioned passive avoidance reflex» (CPAR) in conjunction with the corticosterone level in the blood and the dynamics of changes in the content of noradrenaline in the dorsal hippocampus on the background of immobilization load of stress.
Methods
The experiments were carried out on male Wistar rats weighing 250-300g, contained in standard vivarium conditions with free access to food and wa-
ter (illumination mode - 8.00-20.00). The rats were obtained from the "Stolbovaya" branch of the Scientific Center for Biomedical Technologies of the Federal Medical and Biological Agency. After a week of adapting rats to vivarium conditions, they were tested in an open field. The level of motor and research activity of rats was assessed by the total activity criterion calculated as the sum of the central and peripheral squares intersected by animals, the investigated objects and stands in the central and peripheral zones of the open field. Values of the total activity criterion of less than 80 characterized the behavior of the rat as passive, and values above 120 - as active. Rats with average activity rates were excluded from further experiments.
The total number of rats in the experiment was 70 individuals. According to the results of the study in the open field test, 35 rats were assigned to a group of behaviorally active rats, 35 rats - to a group of passive animals. The active and passive individuals were divided into five subgroups of seven animals in each: the animals in the first and second subgroups were examined for the behavior in the CPAR test; in rats of the third and fourth subgroups, the content of corticosterone in the blood was studied; in the rats of the fifth subgroup, the noradrenaline content profile in the hippocampus was assessed dynamics of stress load. The rats of the first and third subgroups were not subjected to stress loading, animals of the second, fourth and fifth subgroups were subjected to stress loading.
The stress load was modeled by fixing the rats on the platform for the paws for 1 hour with simul-
taneous stochastic electrocutaneous stimulation of 0.25 mA, 50 Hz, with a duration of 1 minute and intervals of 3 to 8 minutes.
The experimental chamber of the CPAR test consisted of two compartments: the illuminated open and the dark one closed. Between the compartments there was a door. The darkened compartment was equipped with an electrically conductive floor. The rat was placed in a lighted compartment. The time of the animal's stay in the illuminated compartment before opening the door was 10 seconds. After opening the door, the latent period of the animal's transition into the dark compartment was measured. After the transition of the rat to the dark compartment, an electrocutaneous stimulation (ECS) was applied to the electroconductive field for 5 seconds with a current strength of 1.5 mA, a frequency of 1 Hz, and a pulse duration of 100 ms. The first presentation of the CPAR test ended after the rat emerged from the dark compartment or 10 seconds after the end of the ECS. Repeated testing of CPAR was carried out 72 hours after the first. During the repeated testing of CPAR, the latent period of entering into the dark compartment was evaluated. Control rats were tested without a stress load. The rats of the test groups were subjected to an hourly immobilization stress loading two hours before the repeated testing of CPAR.
The content of corticosterone was studied in blood obtained by decapitation of rats. Decapitation was performed on unanesthetized animals for a time not exceeding 20 seconds. The content of corticosterone was determined by enzyme immunoassay using the rat corticosterone kit (Immu-nodiagnostic Systems Ltd, AC-14F1).
The content of noradrenaline in the dorsal hippocampus was determined by microdialysis followed by high-performance liquid chromatog-raphy with electrochemical detection. Operations to implant microdialysis probes in the dorsal hippocampus were performed under chloral hydrate anesthesia (intraperitoneally, 400 mg/kg). The coordinates of the dorsal hippocampus in accordance with the atlas [17] were AP = 4.6 mm, Lat = 2.0 mm, Vent = 4.6 mm from bregma. Concentric probes CMA 12 (CMA Microdialysis Sweden) with a pore size of 20 kD were used. Dialysis was collected 48 hours after the implantation of the cannulae. The collection time of each dialysate was 20 min. Before the experiment, the probes were perfused with an artificial cerebrospinal fluid for 2 hours. The scheme of the experiment included a sequential collection of eight dialysates.
The first two dialysates were obtained in rats in the initial state of rest. At the beginning of collection of the third dialysate, the animals were fixed on a platform. During the stress load, the 3rd, 4th and 5th dialysates were collected. At the end of stress after the animals were released, the 6th, 7th and 8th dialyzates were collected. The content
of noradrenaline in dialysates is expressed as a percentage of its content in the two initial dialyzates. After the end of the experiments, the rats were decapitated, the brain was removed and frozen for subsequent histological examination of the position of the probes in the dorsal hippocampus. To check the position of the probes in the dorsal hippocampus, sections with a thickness of 20 ^m were made on a freezing cryotome and stained with a cresyl violet. The results of noradrenaline content were obtained in rats with confirmed location of the microdialysis probe in the dorsal hippocampus.
The concentration of noradrenaline in dialyzates was measured by high performance liquid chroma-tography using an LC-304T chromatograph (BAS, West Lafayette, USA) equipped with a Rheodyne 7725 injector with a 50 |jl loop to sample. The materials studied were separated on a reversed-phase Reprorsil C18 column (3 ^m, 2 * 100 mm). The electrochemical determination of the substances was carried out on an LC-4B amperometric detector with a TL-5 cell (BAS, WestLafayette, USA) at a potential of +0.65 V against the Ag / AgCl reference electrode.
The results of the experiments are presented as mean ± standard error of the mean. Statistical processing of the results was carried out using the program Statistica 8.0 (StatSoft, Inc). The reliability of the differences was evaluated after checking the normality of the data distribution. With the normal distribution of data, the reliability of the differences was assessed using the Student's parametric t-test. By an abnormal distribution, variance analysis was used to assess the reliability of the differences. With its help, the factors that significantly influenced the differences were identified, then the groups were compared in pairs using non-parametric Mann-Whitney criteria in the case of comparing dependent populations or the Wil-coxon test when comparing independent groups.
The experiments were carried out in accordance with: the requirements of orders No. 1179 of the Ministry of Health of the USSR (11.10.1983) and No. 267 of the Ministry of Health of the Russian Federation (19.06.2003), "Rules for conducting studies using experimental animals" of P.K. Anokhin Research Institute of Normal Physiology of the Russian Academy of Medical Sciences (protocol No. 1 of 3.09.2005) and "Rules for the treatment, maintenance, anesthesia and killing of experimental animals".
Results
The results of the study of the rat behavior in the "open field" test are presented in Table. 1. As shown by the results of the testing, the groups of active and passive rats isolated from the general population differed significantly in the number of crossed peripheral and central squares,
in the number of stands in the peripheral and central zones, in the number of investigated objects and in the duration of grooming (Table 1). Thus, according to the results of testing the rats in the "open field" test, groups of active (n = 35) and passive (n =
35) individuals with a highly significant (p <0.001) difference in the total criterion of motor and research activity were isolated from the general population of 150 rats (Table 1).
Table 1
Rat behavior in the "open field" test
Indicator active (n=14) passive (n=14)
latent period of the first movement 1,75±0,30 2,71±0,41
latent period of access to the central zone 61,44±7,11 62,55±10,04
number of intersected peripheral squares 98,63±2,41 56,45±1,64***
number of intersected central squares 10,28±1,34 3,90±0,55***
number of stands in the peripheral zone 17,47±1,28 11,26±0,82***
number of stands in the central zone 0,44±0,12 0,06±0,04***
number of objects examined 3,84±0,59 2,13±0,35*
duration of grooming 8,91±1,46 18,71±1,96***
number of fecal boluses 4,03±1,38 3,58±1,34
amount of urination 0,38±0,10 0,71±0,15
SAA 160,65±1,86 73,81±1,45***
Note: * - p <0.05 and *** - p <0.001 between groups of active and passive rats.
The results of experiments on the behavior of rats in the CPAR test are presented in Figure 1. Analysis of the results with the help of variance analysis revealed a significant effect of behavioral activity (F (1.52) = 27.2833, p <0.001) and repeated
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measurements in the CPAR test (F (1.52) = 14.6724, p <0.001). The value of the latent period of entry into the dark compartment in behaviorally passive rats was significantly higher in the second testing of the CPAR compared to the value in the first
1A
Figure 1. The values of the latent periods of rats entering the dark compartment in the test of the CPAR at the first (light columns) and the second (shaded columns) tests of active (1) and passive (2) rats not stressed (A) and against the background of the stress load before the second testing (B). * - p <0.05 compared to the values at the first testing of the CPAR of passive rats; # - p <0.05 compared with the values for the second test of active rats; + - p <0.05 compared with the values for the second test of active rats without stress loading.
1A: 37,86±9,33,17,71±3,21 2A: 50,86±12,04, 134,14±23,66 1B: 47,00±15,65, 33,43±5,18 2B: 34,57±6,73 173,57±3,12
test in both non-stimulated (p <0.05) and strained passive individuals (p <0.05). Also, in passive rats, the latency period of entering the dark compartment during the second test significantly exceeded the latent period of approach in active animals, both at the first and second presentation of the test (p <0.05). In the second test, the behaviorally active, stressed rats entered the dark compartment after a significantly longer period of time compared to the behaviorally unstressed animals (p <0.05) (Fig. 1). Behaviorally active, stressed rats entered the dark compartment where they had been presented with an ECS with a latency period equivalent to the period of the first presentation of the CPAR (Figure 1) in the second testing of the CPAR.
The results of the study of the content of corticosterone in the blood of rats are presented in Figure 2. As the results of the conducted experiments show,
the content of corticosterone in the blood of behav-iorally active rats one hour after the end of the stress load is 53.75 ± 4.12 ng/ml, which is significantly higher in comparison with the content in the blood of control active rats not subjected to a stress load of 37.76 ± 4.03 ng/ml (F (1.12) = 7.6874, p <0.05). The level of corticosterone in the blood of passive rats not subjected to stress loading was 45.48 ± 4.29 ng/ml. This was significantly inadequate in comparison with the level of behaviorally active non-stressed rats (F (1.12) = 1.7189, p = 0.21437). The content of corticosterone in the blood of behaviorally passive rats after a stress load was 49.61 ± 3.98 ng/ ml. This was slightly higher than the concentration of non-stimulated individuals and was significantly less than the level in the behaviorally active rats after the stress load (Figure 2).
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Figure 2. The content of corticosterone in the blood of active (1) and passive (2) rats not subjected to stress loading (light columns) and an hour after the hour of immobilization load (shaded columns). * - p <0.05 in comparison with the content in active non-stimulated rats. 1: 37,76±4,03 53,75±4,12 2: 45,48±4,29 49,61±3,98
In order to study the characteristics of the neuro-chemical profile of the dorsal hippocampus, which performs important functions in the processes of fixation and retrieval of memory [4], in controlling the motor activity [7] and in regulating the activity of the hypothalamic-pituitary-adrenal axis [10], the following series of experiments on the study of noradrenaline content in the dynamics of immobilization stress in behaviorally active and passive rats by means of microdialysis. The results of the experiments are shown in Figure 3. Analysis of experimental results with the help of variance analysis revealed a significant effect of the activity factors (F (1.12) = 16.29, p <0.01) and stress (F (7.84) = 3 , 72, p <0.01). The level of noradrenaline
in the dorsal hippocampus of active rats increased significantly at the onset of immobilization stress and remained significantly higher than the baseline during the stress and subsequent hours (p <0.05). In behaviorally passive rats, the level of noradren-aline in the dorsal hippocampus did not change during the immobilization stress load as compared to the initial one. A significant decrease in the level of noradrenaline in passive rats was found in comparison with the initial one in the post-stress period 40-60 minutes after the end of the hour immobilization (p <0.05). The level of noradrenaline in the hippocampus of active rats was significantly higher when compared with the level in passive animals (p <0.05) (Figure 3).
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Figure 3. Concentration of noradrenaline in dialysates of the dorsal hippocampus collected in the initial resting state, during the hour immobilization and after its termination in active (solid line) and passive (dashed line) rats. * - p <0,05 in comparison with the level of norepinephrine in the initial state of rest; # - p <0.05 compared with the level of passive rats.
-40 min -20 min 0 20 min 40 min 60 min 80 min 100 мин
Act 97,26±10,48 102,74±10,48 254,78±41,68165,78±12,18 200,22±32,06 157,49±23,97 184,79±19,56 211,05±31,08 Pass 89,47±13,15 110,55±13,15 96,59±24,13 112,75±30,28 90,88±15,40 83,70±21,33 103,35±20,64 45,97±6,92
Discussion
In accordance with the views of the scientific school P.K. Anokhin, which was developed in the scientific school of K.V. Sudakova, the achievement of the social or biological result necessary for the organism acts as a system-forming factor that organizes the inclusion and dynamic interaction of the organism's processes ensuring the achievement of an adaptive result [3]. The characteristics of primary cerebral reactions in different subjects determine the individual pattern of the organism's reactions in conditions of conflict situations in which the organism is deprived of the possibility of achieving the required result [2, 5, 12]. When assessing the functional role and physiological orientation of the changes in the content of neurotrans-mitters detected in the experiment, it is necessary to take into account the effectiveness of the behavior [7].
In the analysis of behavior in the CPAR test, the domination of innate biological motivation in the avoidance of aversive bright light and open space irritants is traditionally considered in rats, causing the animals to move into a dark enclosed compartment from the illuminated open compartment. During the second testing of the CPAR for the duration of the latency period of the transition of the animal to the dark compartment, the influence of the avoidance motivation of the ECS is assumed, the memorable trace of which can be formed
as a result of its presentation during the first test. With an increase in the duration of the latent period of transition to the dark compartment, in the second test, in comparison with the first one, an opinion is made about the formation and reproduction of a memorable trace on the ECS and the animal's predominance of avoidance of ECS. In case of equivalence or shortening of the latent period - the absence of the formation or reproduction of a memorable trace, or the lack of dominance in the animal of the motivation to avoid ECS.
We in behaviorally passive rats showed an elongation of the latency period of entering into the dark compartment during the second testing of CPAR as compared with the values at the first testing. In the rats active in behavior, the latency period of entering the dark compartment during the second test did not differ from the period of approach during the first test. This observation can be interpreted as the dominance of the ECS avoidance motivation on the basis of the formed memory trace in behaviorally passive rats and the lack of dominance of the avoidance motivation for ECS in behaviorally active animals.
The elongation of the latent period of entry into the dark compartment during the second testing of CPAR in passive behavior of rats not detected in active animals was also noted in the experiments of other authors. Thus, in the studies of R. Landgraf et al. [14], carried out on the rats select-
ed for the manifestation of anxiety with congenital motor activity, it was noted that there was no lengthening of the latency period of entering into the dark compartment at the second presentation of the CPAR in behaviorally active rats, whereas in passive patients, the increase in the time of entry into the dark compartment was revealed.
It is known that memory is one of the complex forms of brain activity, which includes various components [16]. In the mechanisms of spatial memory an important role is played by the hippocampus [11]. There are experimental data on changes in the emotional or spatial components of memory under various effects, for example, when injecting interleukin 1p [19]. Intra cerebral administration of interleukin 1p worsened spatial memory in the Morris water labyrinth and the eight ray labyrinth, but improved memory in the CPAR test. Separate neurochemical mechanisms of the brain can have a specific effect on various forms of memory. So, in the mechanisms of contextual and spatial memory, noradrenergic processes are most clearly involved, while in the formation and extraction of emotionally significant memories their role seems less significant [11]. There are also experimental data on the relationship of various forms of memory with high or low levels of cortisol in the blood under stressful stress conditions. Thus, in experiments [8] it was noted that persons who had an increase in the level of cortisol in the blood under stressful conditions, reproduced emotionally negatively colored tasks for memory testing worse. At the same time, according to the results of [8], the memory reproduction with emotional coloring improves under stress loads in persons who under stress do not show an increase in the level of cortisol in the blood. These observations are consistent with the results of our study. The best reproduction of an emotionally negatively colored task was observed in behaviorally passive rats that did not show an increase in corticosterone content in the blood after an immobilization stress load.
The elongation of the latent period of entry into the dark compartment during the second testing of the CPAR can be interpreted as the achievement by animals of the result of avoiding the ECS in the dark compartment when implementing the strategy of the passive avoidance behavior of the aversive stimulus, the memorable track of which was formed and reproduced. The implementation of this behavioral strategy in behavioral-ly passive animals is combined with the invariance of the noradrenaline content in the dorsal hippocampus during the immobilization stress and after its termination, as well as with the invariance of the level of corticosterone in the blood after stress. In this case, the active strategy of the inevitability of the aversive stimulus in the CPAR test, which is characteristic for animals active in behavior, is combined with the increase in the content
of noradrenaline in the dorsal hippocampus during and after stress, with an increase in the level of cor-ticosterone in the blood after the end of the stress load and the equivalence of the latent period of entry into Dark compartment during the second testing of the CPAR.
The main source of noradrenaline in various structures of the brain are the projections of neurons of the blue spot in different parts of the brain. The activity of the blue spot neurons is expressed by the influence of the neurons of the paraventric-ular nuclei of the hypothalamus, which regulate the glucocorticoid hormones in the blood. Cor-ticotropine releasing factor stimulates neurons of the blue spot, which is accompanied by an increase in the content of noradrenaline in various parts of the brain [10]. This is consistent with the results obtained in our study. The elevated level of glucocorticoid hormone corticosterone after immobilization stress loading is revealed in behaviorally active rats, the neurochemical profile of the hippocampus in which is characterized by an increase in the level of norepinephrine during and after the stress load.
Thus, the interpretation of the results of the study of animal behavior in the CPAR test requires taking into account the effectiveness of the behavior for an adequate interpretation of the detected indicators. The formation of a memorable trace of aversive electrocutaneous irritation during the first testing of CPAR acts as one of the factors determining the behavior of animals in this test. When interpreting the research results from the viewpoint of the theory of functional systems, it is possible to draw a conclusion about various strategies for the effective behavior of animals in the CPAR test detected on the basis of recording the lengthening of the latent period of entering the dark compartment or the equivalence of the time interval for entering the dark compartment during the first and second tests of the CPAR. Passive behavioral strategy to achieve the result of avoiding the aver-sive stimulus is combined with the invariance of the content of noradrenaline in the dorsal hippocampus during the immobilization stress and after its termination and with the constant level of cor-ticosterone in the blood after stress. The active behavioral strategy of the inevitability of the aversive stimulus is accompanied by an increase in the content of norepinephrine in the dorsal hippocampus during the immobilization stress and after its termination and an increase in the level of corticoste-rone in the blood after stress.
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Contacts
Corresponding author: Umryukhin Alexey Evgenievich, Doctor of Medical Sciences, Head of the Department of Normal Physiology of I.M. Sechenov First Moscow State Medical University, Moscow.
125009, Moscow, ul. Mokhovaya, 11 (4). Tel.: (499) 2480553. E-mail: alum1@yandex.ru
Babayevskaya Diana Ivanovna, worker of the Laboratory of psychiatric neurobiology I.M. Sechenov First Moscow State Medical University, Moscow. 125009, Moscow, ul. Mokhovaya, 11 (4). Tel.: (499) 2480553. E-mail: alum1@yandex.ru
Kalinin Sergey Alekseevich, worker of the Laboratory of psychiatric neurobiology I.M. Sechenov First Moscow State Medical University, Moscow. 125009, Moscow, ul. Mokhovaya, 11 (4). Tel.: (499) 2480553. E-mail: alum1@yandex.ru
Chekmareva Natalya Yurevna, assistance lecturer the Department of Normal Physiology of I.M. Sechenov First Moscow State Medical University, Moscow.
125009, Moscow, ul. Mokhovaya, 11 (4). Tel.: (499) 2480553. E-mail: alum1@yandex.ru
