Neuroscience and Behavioral Physiology, Vol. 35, No. 4, 2005
The Effects of Mexidol on the Acquisition of Food-Related Conditioned Reflexes and Synaptic Ultrastructure in Field Ca1 of the Rat Hippocampus after Single Acoustic Stimuli with Ultrasonic Components T. G. Alekseeva,1 E. V. Loseva,1 and T. A. Mering2*
UDC 612.821
Translated from Zhurnal Vysshei Nervnoi Deyatel’nosti, Vol. 54, No. 2, pp. 269–276, March–April, 2004. Original article submitted August 29, 2002, accepted March 11, 2003. The effects of a complex acoustic signal with ultrasonic components on the ultrastructure of synapses field CA1 of the rat hippocampus were studied in conditions of two-week courses of the wide-spectrum antioxidant Mexidol (compared with an untreated group); the effects of complex acoustic signals on the dynamics of acquisition of a food-related conditioned reflex using a standard stimulus (a tone) and on the acquisition of a trace conditioned reflex to estimating time intervals were also studied, in the same groups of rats. Controls consisted of unstressed rats treated and not treated with Mexidol. Ultrastructural analysis of the redistribution of vesicles in the synaptic terminals of hippocampal field CA1 showed that synaptic transmission was impaired when assessed one day after exposure to the complex acoustic signal. Mexidol prevented impairment of synaptic transmission. The complex acoustic signal had negative effects on conditioned reflex activity in rats and Mexidol had normalizing actions on the acquisition of conditioned reflexes in stressed rats. These results lead to the conclusion that the antioxidant Mexidol can be applied to the prophylaxis of the impairments in CNS cognitive functions frequently seen in stress. KEY WORDS: acoustic stress, food-related conditioned reflexes, hippocampus, synapse ultrastructure, the antioxidant Mexidol.
Sounds of different frequencies and intensities play an important role in the lives of animals and humans. Sound can warn of danger and attract attention, and can also be the source of a stress-like state, leading to inadequate behavior. Signals with ultrasonic components are of particular importance in the lives of many mammals, including rats. Ultrasound with a particular spectrum of frequencies increases anxiety and promotes panic in rats, and they tend to avoid signals of this type [23]. This phenomenon is used for preparing devices for repelling rodents. Previous studies have shown that exposure to transient complex acoustic signals (CAS) with ultrasonic compo-
nents (the jangling of keys) produces audiogenic convulsions in 40–60% of Wistar rats [7, 9, 28]. It would seem that this type of signal is a stressor and can impair both the structure and function of the CNS. Ultrafine analysis of the state of synapses led to descriptions of disturbances in synaptic transmission on exposure to stress [8, 33]. There are numerous data on impairments of cognitive functions in stresses of various types [24–26]. However, we were unable to find any studies describing the effects of acute acoustic stimuli on the ultrastructure of the hippocampus. We suggested that the action of CAS may also disturb synaptic transmission in the hippocampus and the cognitive functions of the brain, particularly the acquisition of food-related conditioned reflexes. Activation of lipid peroxidation is known to be a common component of stress impairments [12]. The occurrence of these processes in the post-stress period is accompanied by activation of the mechanisms of natural antiradical
1 Institute
of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences. 2 Institute of the Brain, Russian Academy of Medical Sciences, Moscow; e-mail:
[email protected]. * Deceased.
363 0097-0549/05/3504-0363 ©2005 Springer Science+Business Media, Inc.
364 defense. The effects of natural antiradical defense functions can be enhanced by exposure to antioxidant agents. If our suggestion regarding the negative influences of CAS on CNS structure and function is correct, then it should be possible to prevent these changes using antioxidants. Mexidol is regarded as one of the most effective antioxidants – this is a Russian agent with a wide spectrum of action, including antistress effects [13, 19]. Many reports have described studies of the properties of Mexidol, though its actions at the ultrastructural level remain poorly investigated. We suggested that Mexidol might be used for the prophylaxis of the negative sequelae of CAS at the structural and functional levels. The aims of the present work were as follows. 1. To study the effects of CAS on the ultrastructure of synapses in hippocampal field CA1 in rats with and without treatment with Mexidol. 2. To study the effects of CAS on the dynamics of the acquisition of a food-related conditioned reflex to a normal stimulus (a tone) and the acquisition of a food-related trace reflex to estimated time intervals in the same groups of rats. 3. To study these measures in unstressed rats with and without Mexidol.
METHODS Studies were performed using adult female Wistar rats weighing 180–250 g, from the Stolbovaya supplier. Animals (n = 56) were divided into four groups. Rats of group 1 (“stress”, n = 15) were placed in a chamber for 90 sec and exposed to complex acoustic signals with multiple peaks in the frequency range 13–85 kHz with a peak at 20–40 kHz and a mean intensity of 50–60 dB, using a standard method. Animals of group 2 were subjected to the same stimulation, but after prior (7 days) treatment with Mexidol (the “Mexidol + stress” group, n = 14). Controls consisted of intact animals (the “normal” group, n = 15) or animals given Mexidol (the “Mexidol” group, n = 12), kept in the same chamber but without exposure to CAS. Mexidol was dissolved in the drinking water (100 mg/150 ml) provided for one week before and six days after exposure to CAS. Electron microscopic studies of synapses were performed one day after exposure to CAS. These studies were undertaken in 19 rats: from the “normal” group (n = 5), the “stress” group (n = 5), the “Mexidol + stress” group (n = 4), and the “Mexidol” group (n = 5). Experimental animals and controls were sacrificed one day after exposure to CAS. Brains were removed rapidly and the frontal zone was removed at the level of the dorsal hippocampus and material was placed in 2.5% glutaraldehyde solution in phosphate buffer for 15 min. An MBS-9 microscope was used to cut blocks of tissue from field CA1. Blocks were then cut into small fragments such that the long axis included all layers of the hippocampus. Specimens were fixed for 4 h in 1%
Alekseeva, Loseva, and Mering osmium tetroxide solution in phosphate buffer as described by Palade [31]; this was followed by dehydration in alcohols of increasing concentrations and contrasting with 3% uranyl acetate in 100° alcohol. Samples were then placed in acetone for 10 min and embedded in Araldite. Parallelplane embedding allowed orientation of specimens such that the pyramid could narrow along its long axis. Ultrathin sections (500 µm) were prepared on an LKB-3 ultratome. Section areas corresponded to the surface areas of specimens along the long axis. Osmium sections were counterstained with lead citrate for 0.5 h [32] and examined using a Phillips electron microscope (Austria). Sections from each rat were examined at a magnification of ×66,000 using two grids, one from each of two blocks. Cross sections of synapses having an active zone and synaptic vesicles were selected. Only axodendritic and axospinous contacts were examined. Each terminal was assessed in points (1–4) in terms of two features: 1) the distribution of synaptic vesicles (1 point indicated that all vesicles were concentrated in the active zone; 2 points indicated that some vesicles were located in the active zone, 3 points indicated that vesicles were located uniformly across the area of the terminal cross section, and 4 points indicated that vesicles were concentrated in the center of the terminal); 2) the proportion of the terminal cross section filled with vesicles (1 point = 25%; 2 points = 50%; 3 points = 75%, 4 points = 100%). A visual rank classification of synapses was performed in terms of morphological features [10]. Totals of 50 synaptic terminals were assessed in terms of the two characteristics in each rat. Data were recorded and analyzed statistically as follows. Data for each synapse were entered into a matrix (5 × 10) for each feature. For convenience, each matrix was divided into groups of 10 synapses and the numbers of synapses with 1, 2, 3, and 4 points were counted for each 10 synapses. There were five groups of 10 neurons for each rat and 25 or 20 neurons for each group (20 for the “Mexidol + stress” group). This yielded variation series consisting of 25 (20) values or each point for one sign. Further statistical processing was performed using the Statistica computer program to run the t test for independent variables. Acquisition of food-related conditioned reflexes to a standard stimulus (a tone at 40 dB) and to estimates of time intervals was developed as described by Mering [13]. These experiments used 37 animals: a “normal” group (n = 10), a “stress” group (n = 10), a “Mexidol + stress” group (n = 10), and a “Mexidol” group (n = 7). After exposure to CAS and for the next five days, all animals underwent development of a food-related conditioned reflex to the tone (20 combinations per experiment). This was performed in a chamber (30 × 40 × 30 cm) with an opening (diameter 4 cm) in the wall (30 × 30 cm) at a height of 7 cm from the floor. A platform (10 × 7 × 5 cm) was placed on the inside of this wall; by standing on the platform rats could obtain food through
The Effects of Mexidol on the Acquisition of Food-Related Conditioned Reflexes the opening. The tone was presented with a strictly stereotypical temporal pattern with an interval of 60 sec. On exposure to the tone for 15 sec, the rats had to mount the platform and take food (spheres of bread in milk) through the opening in the chamber wall. Protocols were used to select traces of the acquisition of the food-related conditioned reflex to the standard stimulus (the tone) for analysis of the following parameters: washing and freezing times, which characterize the balance of inhibitory and excitatory processes in the CNS; the number of intersignal reactions, which reflects both locomotor activity and the extent of food motivation; the number of correct decisions, i.e., timely approaches to the feeder and taking of food, which characterizes food motivation and understanding of the task; the time spent sitting by the feeder not associated with taking food, which is a complex measure reflecting both the level of understanding of the task and the presence of food motivation. In combination with the other parameters, increases in this measure may be evidence either for inhibitory processes in the CNS or, conversely, hyperactivity and powerful food motivation. Food takes beyond the first 15 sec were disallowed. After exposure to the tone 100 times, it was removed and, during one experiment, the same animals were used to test the ability to acquire a conditioned reflex to their estimate of the time interval. Responses were considered correct when the rats mounted the platform 60 ± 10 sec after the previous food take and showed no more than one intersignal excursion. During the session, rats were presented with this time interval 15 times. The proportion of correct responses (%) was measured. Statistical comparisons between all groups of rats for all measures were based on the t test for independent variables using the computer program Statistica.
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Fig. 1. Distribution of synaptic vesicles in different parts of terminals in normal conditions and after various treatments. The vertical axis shows the proportion of terminals with different extents of this characteristic compared to the total number of synapses analyzed (250), %. *Significant differences compared with the “normal” group; #significant differences compared with the “stress” group (p values are given in the text).
RESULTS Electron Microscopic Studies of Synapses in Hippocampal Field CA1. Data on the distribution of vesicles in synaptic terminals and the proportion of the cross-sectional area of the terminals filled with vesicles in hippocampal field CA1 from rats of all groups are plotted as histograms (Figs. 1 and 2). The results are presented as percentage ratios of the numbers of synapses with different extents of these characteristics to the total numbers of terminals examined. Analysis of data on the distribution of vesicles in synaptic terminals (Fig. 1) showed that presentation of CAS was followed one day later by the detection of differences between groups at the ultrastructural level. Thus, the number of synapses with vesicles concentrated close to the active zone was significantly larger in the “Mexidol” group than in normal controls (p = 0.014), while the “Mexidol + stress” group had more such synapses than the “normal” group (p = 0.0007) and stressed animals (p = 0.0073).
Fig. 2. Group differences in the proportions of the cross-sectional areas of terminals filled with synaptic vesicles in normal conditions and after various treatments. The vertical axis shows the proportion (%) of terminals with different extents of this characteristic in relation to the total number of synapses analyzed (250); the horizontal axis shows the proportion of the cross-sectional area of terminals filled with synaptic vesicles, %. *Significant differences compared with the “normal” group; #significant differences compared with the “stress” group; +significant differences compared with the “Mexidol + stress” group (p values are shown in the text).
Synapses with vesicles partially located close to the active zone were found in the stress-free group treated with Mexidol statistically more often than in stressed rats (p = 0.011) and slightly more often than in the other groups. Uniformly distributed vesicles were found statistically significantly less frequently in the synapses of rat hippocampi
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Fig. 3. Group differences in the dynamics of the duration of non-specific responses – washing and freezing not associated with conditioned reflex activity in animals during acquisition of a conditioned reflex to a tone. The vertical axis shows the proportion of the duration (%) spent washing and freezing in relation to the duration of a single experimental session (20 min); #significant differences compared with the “stress” group (p values are given in the text).
Fig. 4. Group differences in the dynamics of the time spent sitting by the feeder not associated with food consumption in animals during acquisition of a conditioned reflex to a tone. The vertical axis shows the time spent sitting by the feeder not associated with food consumption, sec. *Significant differences compared with the “normal” group; #significant differences compared with the “stress” group; +significant differences compared with the “Mexidol + stress” group (p values are given in the text).
after stress with Mexidol treatment and in stress-free Mexidoltreated animals than in the “normal” group (p = 0.042 and 0.012 respectively). These synapses were found rather less often in stressed rats than in the “normal” group. Synapses with vesicles distributed centrally were typical of stressed animals. Thus, as compared with this
Alekseeva, Loseva, and Mering group, the numbers of these synapses in the “Mexidol,” “Mexidol + stress,” and “normal” groups were statistically significantly less frequent (p = 0.0073, p = 0.025, and p = 0.0024 respectively). The proportions of the cross-sectional areas of terminals filled with vesicles (Fig. 2) were also different in different groups of animals. Synapses 25% filled with vesicles were statistically significantly more common in rats of the “Mexidol” group than in stressed rats and animals of the “Mexidol + stress” group (p = 0.0057 and p = 0.048 respectively) and slightly more often than in the “normal” group. However, terminals 50% filled with vesicles were significantly less common in this group than in the others. The proportions of rats of the different groups with synapses 75% filled with vesicles were not statistically significantly different from each other. The proportion of synapses 100% filled with vesicles was statistically significantly greater in stressed rats than in nonstressed rats given Mexidol (p = 0.0079) and slightly greater than in the other groups. Interindividual differences were insignificant. Analysis of the state of synaptic terminals showed that stressed animals underwent a redistribution of vesicles towards the centers of terminals as compared with normal animals, along with an increase in the cross-sectional areas of terminals filled with synaptic vesicles. This may be evidence for increased transmitter synthesis and, on the other hand, for the inability to utilize excess numbers of vesicles. At the same time, it appears that exposure to CAS disturbs biochemical processes associated with transport of vesicles to the active zone. Acquisition of Conditioned Reflexes. The following parameters were assessed on a day-by-day basis: the total time (sec) of washing and freezing; the number of intersignal excursions; the number of correct responses; the time (sec) of sitting on the box with the nose facing the feeder when not directly associated with food consumption. Analysis of the dynamics over 5 days of measures of the acquisition of the food-related conditioned reflex to the tone showed that acquisition of this reflex in different groups of rats occurred according to different stereotypes. Thus, the washing and freezing times during experimental sessions in normal rats showed insignificant variation on all experimental days (Fig. 3). On the third day after stress, this measure was significantly increased (p < 0.01) compared with the “Mexidol + stress” group, normalizing only by day 5. Rats of the “Mexidol” and “Mexidol + stress” groups were no different from normal in terms of this measure. Analysis of the numbers of intersignal reactions showed that the dynamics of this measure after stress in Mexidoltreated animals were similar to the dynamics in normal animals. At the same time, rats of the “stress” group showed significant reductions (p < 0.01) on days 1 and 3 than in the “Mexidol” group, and showed fewer of these reactions than normal rats on day 2. In unstressed animals treated with
The Effects of Mexidol on the Acquisition of Food-Related Conditioned Reflexes
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ly lower than in animals of the other groups (p = 0.03 compared with the “normal” group, p = 0.0001 compared with the “Mexidol + stress” group, p = 0.02 compared with the “Mexidol” group). There were more correct responses in rats of the “Mexidol + stress” (61.6%) than in the “normal” and “Mexidol” groups.
DISCUSSION
Fig. 5. Group differences in the numbers of correct responses in animals during acquisition of a conditioned reflex to an estimated time interval. The vertical axis shows the proportion of correct responses (%) in relation to the total number of combinations in the experiment (15). #Significant differences compared with the “stress” group (p values are given in the text).
Mexidol, this measure was slightly higher than in all other groups of animals, approaching normal only by day 4. Analysis of the numbers of correct responses, i.e., correctly timed approaches to the feeder and taking of food, showed that the dynamics of this measure were virtually identical in the “normal” and “Mexidol + stress” groups. However, stressed animals showed a tendency to a decreased number of correct responses on experimental days 1 and 2. Rats of the “Mexidol” group, conversely, showed a significant increase in this measure compared with the “stress” and “Mexidol + stress” groups in the first two days (p = 0.032 compared with the “Mexidol + stress” group on day 1; p = 0.00099 and p = 0.0114 compared with the “stress” group on days 1 and 2 respectively). The times spent sitting by the feeder, not associated with food consumption, were virtually identical in normal and stressed rats on each experimental day, while this measure was statistically significantly (p < 0.01) greater in stressed rats on day 3 than in the other groups (Fig. 4). At the same time, the time spent sitting by the feeder was statistically significantly greater in stress-free, Mexidol-treated rats than in all other groups on experimental day 1 (p = 0.009 compared with the “Mexidol + stress” group, p = 0.00066 compared with the “stress” group, p = 0.004 compared with the normal group) and was greater than in stressed, Mexidoltreated rats on day 2 (p = 0.01). Data obtained from testing the ability to acquire the timeestimation conditioned reflex (Fig. 5) showed that the proportion of correct responses in normal animals was 45.8%, which was comparable to the level seen in the “Mexidol” group (46.6%). At the same time, the number of correct responses in rats of the “stress” group (21.5%) was statistically significant-
Thus, our experiments performed at the ultrastructural and systems (behavioral) levels demonstrated that there is potential for using Mexidol for prophylaxis of stress-like states induced by sound stressors. Mexidol improved the efficiency of synaptic transmission and thus appears to have prevented disturbances in cognitive functions. However, it should be noted that unnecessary prolonged use of the antioxidant Mexidol when sharp changes in homeostatic equilibrium in the body are not foreseen may lead to negative sequelae, particularly inappropriate overexcitation of the CNS. Our results support and supplement published data on disruptions of synaptic transmission in synapses in fields CA1 and CA3 of the rat hippocampus subjected to a variety of stressful treatments [8, 27, 29, 30, 33–35]. Published data provide evidence for particular changes in the fine structure of the hippocampus in response to various types of stressors. Chronic stress is followed by increases in the numbers of spines, mitochondria, and synaptic vesicles, i.e., adaptive processes take place [27, 29, 30]. At the same time, changes were not so marked after acute stress, and affected mainly the synaptic apparatus; in particular, there was a redistribution of vesicles, which appears to be associated with impairment of synaptic transmission [8]. The hippocampus is a structure subject to marked ultrastructural changes due to a variety of treatments (irradiation [22], stress). In other words, the impairments seen here to result from stress and Mexidol-induced changes in synaptic transmission in the hippocampus can impair or alter the learning process. Normalization of synaptic transmission in animals of the “stress + Mexidol” group, impairments of the latter in animals of the “stress” group, and even the element of hyperactivation of synaptic transmission in rats of the “Mexidol” group were reflected in the animals’ learning. It is likely that changes at the ultrastructural level were quite prolonged, as differences in behavior from normal animals were observed throughout the experimental period. Free radicals are constantly being generated in the body, which also has natural systems for antioxidant defense. Lipid peroxidation processes have deep physiological importance, as in the normally functioning body they maintain the balance of cell division processes (mitosis) and natural cell death (apoptosis). This balance is disturbed in stress conditions, in which the formation of free radicals, leading to damage to cells and their ultrastructure, becomes
368 predominant. Activation of lipid peroxidation is a common component of a variety of stressful treatments [5, 12] and leads to the accumulation of free radicals which disrupt the permeability of cell membranes and cause damage to cell organelles. Lipid peroxidation processes in the post-stress period are accompanied by activation of the mechanisms of natural antiradical defense [1]. The effects of natural antiradical defense can be enhanced and impairments of cognitive function can be prevented using living tissues (for example, neurotransplants of embryonic tissue from the rat amygdala [4, 6]) as well as synthetic antioxidant agents [19]. Mexidol has marked antioxidant properties [21]. The action of Mexidol during aging and brain traumas in rats has been shown to extend the period of active life, to decrease various stress states, and to improve the acquisition and state of conditioned reflexes of different levels of complexity [14, 15]. Courses of Mexidol in aged, emotionally reactive rats with neurosis-like states arising during the acquisition of temporospatial differentiation improved impaired autonomic measures [13, 16]. These data correlated with the recovery of the ultrastructural lesions to neurons and interneuronal connections which accompanied the acquisition of temporospatial differentiation in aged animals. Use of Mexidol before marked irreversible morphological changes delayed the development of pathology and facilitated the occurrence of compensatory-restorative processes [14]. The action of Mexidol is based on the following mechanisms: inhibition of lipid peroxidation processes, stabilization of biological membranes, activation of the energy functions of mitochondria, and modulation of the functioning of receptor complexes [2, 3, 11, 21]. Mexidol is already used in clinical practice in ischemia and hypoxia as an antiamnestic, antiparkinsonism, anxiolytic, nootropic, antialcoholic, and anticonvulsive agent [2, 14, 17, 18, 20]. From these points of view, the use of Mexidol for prophylactic purposes is justified, as prior to stress it removes free radicals formed in the body, such that there are virtually no free radicals at the onset of stress. Free radicals formed after stress are removed both by natural mechanisms and by Mexidol. As a result, damage to tissue structures hardly occurs and, as a consequence, the stress reaction is decreased or fails to develop. On the other hand, prolonged artificial inhibition of lipid peroxidation processes should prevent apoptotic cell death and increase cell division, i.e., activate various energetic and synthetic processes in the body. From this point of view we can explain the hyperactivity and increased food motivation seen in the present experiments in rats subjected to prolonged Mexidol treatment but not subjected to the acoustic stressor. It would appear that during the observation period, i.e., during the 12 days of Mexidol treatment, we were able to detect rearrangements in rats only at the level of activation of physiological and structural processes.
Alekseeva, Loseva, and Mering CONCLUSIONS 1. One day after exposure to a complex acoustic signal, the hippocampus showed decreases in the efficiency of synaptic transmission, which were manifest as a greater concentration of synaptic vesicles in the centers of large numbers of synaptic terminals than in normal conditions. 2. After treatment with Mexidol, the efficiency of synaptic transmission in the hippocampus was enhanced, which was expressed as an increase in the concentration of vesicles in the active zones of synapses after exposure to complex acoustic signals and a decrease in the number of vesicles in synapses without exposure to complex acoustic signals. 3. The stereotypical dynamics of the acquisition of a food-related conditioned reflex to a tone after exposure to a complex acoustic signal showed differences from normal in some parameters. After treatment with Mexidol, the dynamics of the acquisition of the conditioned reflex in stressed rats was no different from normal but followed the stereotype seen in unstressed animals. 4. Acquisition of the trace conditioned reflex to estimates of time intervals in rats was impaired after exposure to the complex acoustic signal. This ability was no different from normal in rats treated with Mexidol. 5. The results of these studies lead to the conclusion that there is potential for the use of the antioxidant Mexidol for the prophylaxis of impairments induced by acute acoustic exposures.
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