Behavioral phenotyping of mice deficient for tumor necrosis factor

The tumor necrosis factor (TNF) is a cytokine exerting both homeostatic and pathophysiological roles in the central nervous system (CNS). It has been demonstrated that TNF plays roles in such diseases as Alzheimer’s disease, Parkinson’s disease and multiple sclerosis. Nevertheless, the role of TNF in the CNS under normal physiological conditions is poorly studied. A novel mouse strain with TNF deficiency (TNFKO) was developed in S.A. Nedospa­sov’s labora­tory of Engelhardt Institute of Molecular Bio­logy of the Russian Academy of Sciences on the C57Bl/6 (WT) background. In our study, we compared the behavior of TNFKO and WT adult mice in a battery of tests: open-field, elevated plus-maze and the forced-swim test. We showed that TNF deficiency had no effect on locomotor activity or exploration in the openfield test. At the same time, in this test, TNFKO mice spent more time in the center of the arena, but had a higher level of defecation and lower rearing duration. This result indicates that, in the openfield conditions, TNFKO mice show disorientation rather than anxiety-like behavior. There were no differences between TNFKO and WT in anxiety level in the elevated plus-maze test or in depressive-like behavior in the forcedswim test. These data suggest that TNF deficiency leads to changes in neurofunctional interactions that alter the mouse response to mild stress in the open-field test

Effect of Zbtb33 gene knockout and bacterial lipopolysaccharide on home cage behavior in mice

The Zbtb33 gene encodes the bimodal transcriptional repressor Kaiso, which causes epigenetic repression of genes by binding to methylated mCpG islets in the promoters of the genes. Despite the fact that Kaiso is intensively expressed in the central nervous system, its participation in the regulation of behavior is still poorly understood. Only the participation of Kaiso in the regulation of the behavioral response to emotional stress in the open field and forced swimming tests has been shown. The aim of this study is to elucidate the role that Kaiso plays in regulating daily activity, as well as the behavioral response to stimulation of nonspecific immunity. Experiments were performed on adult male mice with Zbtb33 gene knockout (KO) and animals of the C57BL/6 line (wild type, WT). All animals were 11 weeks old, weighed 26 ± 1 g and had SPF (specific pathogen free) status throughout the experiment. The animals of each genotype were divided into three weighted groups of 8 animals each. Initially, the daily dynamics of motor activity, sleep, food and water intake of intact animals was measured using the PhenoMaster software-hardware complex. The animals of each group were then injected with saline (control), 0.1 or 1.0 mg/kg of bacterial lipopolysaccharide (LPS) dissolved in saline, and again measured for their daily activity, food and water intake. Intact KO and WT mice did not differ in the average daily motor activity and sleep duration. However, intact KO mice were less active in the dark time, and also consumed less food and water as compared to intact WT animals. LPS at both doses suppressed motor activity, prolonged sleep duration and caused anorexia in mice of both genotypes. However, the effect of low dose of LPS (0.1 mg/kg) on the food and water intake was more pronounced in KO mice than in WT animals. The results shed light on the biological significance of the Kaiso gene and serve as a justification for the necessity of the normal functioning of this gene in natural populations

Effect of lethal yellow (AY) mutation and photoperiod alterations on mouse behavior

Decrease in natural illumination in fall/winter months causes depressive-like seasonal affective disorders in vulnerable individuals. Obesity is another risk factor of depression. The lethal yellow (AY) mutation causes ectopic expression of agouti protein in the brain. Mice heterozygous for AY mutation (AY/a) are obese compared to their wild-type littermates (a/a). The main aims of the study were to investigate the effects of AY mutation, photoperiod and the interaction between these factors on daily activity dynamics, feeding, locomotor and exploratory activities, anxiety-related and depressive-like behaviors in mild stress condition. Six weeks old mouse males of AY/a and a/a lines were divided into four groups eight animals each and exposed to long- (14 h light and 10 h darkness) or short- (4 h light and 20 h darkness) day conditions for 28 days. Then the behavior of these mice was successively investigated in the home cage, open field, elevated plus-maze and forced swim tests. We did not observed any effect of AY mutation on the general activity, water and food consumption in the home cage; locomotion and exploration in the open field test; anxiety-related behavior in the open field and elevated plus-maze tests. At the same time, AY mutation increased depressive-like immobility time in the forced swim test (F1.28 = 20.03, p = 0.00012). Shortday conditions decreased nocturnal activity in the home cage, as well as locomotion (F1.28 = 16.33, p = 0.0004) and exploration (F1.28 = 16.24, p < 0.0004) in the open field test. Moreover, short-day exposition decreased time spent in the center of the open field (F1.28 = 6.57, p = 0.016) and in the open arms of the elevated plus-maze (F1.28 = 12.08, p = 0.0017) tests and increased immobility time in the forced swim test (F1.28 = 9.95, p = 0.0038). However, no effect of the interaction between AY mutation and photoperiod on immobility time in the forced swim test was observed. Therefore, short-day photoperiod and AY mutation increased depressive-like behavior in the forced swim test by means of different mechanisms

Conducting and automating the water Morris maze test in SPF conditions

The water Morris maze is the basic test to study the spatial ability to learn as well as spatial memory in laboratory rodents. It is a part of a series of tests necessary for behavioral phenotyping of mutant and transgenic mice. At the same time, conducting this test in SPF conditions must comply with very strict regulations concerning pathogen control. A white animal on the surface of whitened water is low contrast and this does not allow the animal to be traced automatically, which represents yet another major problem. A unique installation based on EthoStudio has been developed at the Institute of Cytology and Genetics SB RAS and the Institute of Automation and Electrometry SB RAS. This installation automates the process of tracing mice of any coat color in SPF conditions. This includes a setup to install a plastic water reservoir (110×40 cm), a digital camera and a light source. Water to fill the reservoir was sterilized using a Van Erp Blue Lagoon UV-C Tech 15000 ultraviolet decontaminator. The image of an animal was processed in a frame-by-frame fashion using the EthoStudio program, with the following parameters calculated: latent release time, route covered, cumulative distance to the platform and the time spent in the reservoir sectors. With this installation, we were able to study the spatial ability to learn and spatial memory in mice of the C57BL/6 strain and in mice of the C57BL/6/ Kaiso strain developed on the C57BL/6 background, with the gene encoding the methyl-DNA binding Kaiso protein knocked-out. It has been demonstrated that mice of these strains are able to learn to find the platform in the water Morris maze and have the location of the platform in their memory for at least the next four days

Body composition as an indicator of metabolic changes in mice obtained by <i>in vitro</i> fertilization

To identify body systems subject to epigenetic transformation during in vitro fertilization (IVF), comparative morphological and functional studies were performed on sexually mature offspring of outbred CD1 mice, specific-pathogen-free (SPF), obtained by IVF (experiment) and natural conception (control). The studies included assessment of age-related changes in body weight and composition, energy intake and expenditure, and glucose homeostasis. To level the effects caused by the different number of newborns in the control and in the experiment, the size of the fed litters was halved in the control females. Males obtained using the IVF procedure were superior in body weight compared to control males in all age groups. As was shown by analysis of variance with experiment/control factors, gender, age (7, 10 and 20 weeks), the IVF procedure had a statistically significant and unidirectional effect on body composition. At the same time, IVF offspring outperformed control individuals in relative fat content, but were behind in terms of lean mass. The effect of the interaction of factors was not statistically significant. IVF offspring of both sexes had higher fat to lean mass ratios (FLR). Since adipose tissue contributes significantly less to total energy intake compared to muscle, the main component of lean mass, it is not surprising that at the same level of IVF locomotor activity offspring consumed less food than controls. When converted to one gram of body weight, this difference reached 19 %. One of the consequences of reduced utilization of IVF energy substrates by offspring is a decrease in their tolerance to glucose loading. The integral criterion for the effectiveness of restoring the initial glucose level is the area under the curve (AUC), the value of which was 2.5 (males) and 3.2 (females) times higher in IVF offspring compared to the corresponding control. Thus, the totality of our original and literature data shows an increase in the risk of metabolic disorders in IVF offspring, which is confirmed by epidemiological studies of a relatively young cohort of people born using assisted reproductive technologies

Magnetic resonance spectroscopy of hippocampal and striatal neurometabolites in experimental PTSD rat modeling

The spectrum of the metabolites in the dorsal region of the hippocampus and striatum was studied using the method of 1H magnetic resonance spectroscopy at experimental modeling of the posttraumatic stress disorder syndrome (PTSD) in rats. PTSD was reproduced by exposure of the cat cue to rats daily along 10 day by 10 minutes at once. The anxiety level of animals was estimated 12 days later after the end of the experimental series of stress. Based on the anxiety index, the rats were divided into 3 phenotypes. The animals with an anxiety index &gt; 0.8 (group 1) had lower plasma corticosterone compared with rats form the control group. In animals with an anxiety index in the range 0.7–0.8 (group 2), an elevated corticosterone level was noted. The rats with an anxiety index &lt; 0.7 (group 3) had a lower plasma corticosterone level compared with animals from the control group. Rats of group 2 were characterized by an increased level of GABA in the hippocampus compared with controls. In the remaining groups, the percentages of GABA in the hippocampus and striatum did not differ significantly from the control. The distribution of NAA differed form that of GABA. The highest level of NAA was found in the striatum for rats from group 1, whereas NAA in animals form groups 1 or 3 did not differ from the control. The NAA level in the hippocampus was similar between all groups, including the control. The results obtained indicate that multiple exposures to psychological stress associated with the sense of proximity of a natural enemy in some animals cause an anxiolytic reaction. These animals are characterized by a stable corticosterone level and a stable level of neurometabolites in the studied structures of the brain. For rats with the highest level of anxiety, a lowered level of corticosterone with a constant level of neurometabolites in the hippocampus and striatum is characteristic. And only in rats with an intermediate level of anxiety, synchronization was observed between the increase in plasma corticosterone and the increase in hippocampal GABA content. The results obtained are in good agreement with the ideas of the protective action of glucocorticoids under PTSD manifested in  restraining violations of the psycho-physiological status. The mate rials allow the neurobiological mechanisms of the protective action of glucocorticoids to be detailed

Anxiety and neurometabolite levels in the hippocampus and amygdala after prolonged exposure to predator-scent stress

Here, to study the relationship between anxiety levels with changes in the neurometabolic profile in the hippocampus and amygdala, an experimental predator stress model was reproduced in which Sprague-Dawley rats were exposed to cat urine for 10 minutes on a daily basis for 10 days. At the time of presentation of the stimulus, an online survey of behavioral reactions was conducted. Fear, aggressiveness, avoidance of stimulus and grooming were recorded. Fourteen days after the completion of the last stress exposure, the total level of anxiety was determined in the test of the“cross maze”. Using the method of in vivo NMR spectroscopy, the content of neurometabolites was determined in the hippocampus and in the amygdala. According to the peculiarities of behavioral reactions to a stressor, animals were retrospectively divided into two phenotypes. The first phenotype used a passive behavioral strategy, and the second phenotype was active. In animals of the first phenotype, the indicators of anxiety behavior remained at the control level. In animals of the second phenotype, a decrease in anxiety was observed. Animals of the second phenotype showed elevated levels of lactate in the hippocampus compared to animals of the first phenotype, and the lowest N-acetylaspartate levels significantly differed from those in the control and the first phenotype animals. In the amygdala, in animals of the second phenotype, the content of taurine is sharply reduced in comparison with those in the control and the animals of the first phenotype. Thus, the results obtained indicate a relationship of post-stress changes in anxiety, with the peculiarities of the behavioral reactions presented at the moment of the immediate action of the stressor. Among the hippocampal and amygdala neurometabolites, the most informative for the characterization of the anxiolytic action of the predator stress are identified

Offensive behavior, striatal glutamate metabolites, and limbic–hypothalamic–pituitary–adrenal responses to stress in chronic anxiety

Variations in anxiety-related behavior are associated with individual allostatic set-points in chronically stressed rats. Actively offensive rats with the externalizing indicators of sniffling and climbing the stimulus and material tearing during 10 days of predator scent stress had reduced plasma corticosterone, increased striatal glutamate metabolites, and increased adrenal 11-dehydrocorticosterone content compared to passively defensive rats with the internalizing indicators of freezing and grooming, as well as to controls without any behavioral changes. These findings suggest that rats that display active offensive activity in response to stress develop anxiety associated with decreased allostatic set-points and increased resistance to stress. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.The Russian Science Foundation (grant № 17-15-013418) supported this study. This was supported in part by the contracts of the Ministry of Education and Science of the Russian Federation with South Ural State University (17.7255.2017/8.9) and Institute of Immunology and Physiology (AAAA-A18-118020690020-1). The work was furthermore supported by institutional funds from the State University of New York (SUNY) Upstate Medical University. This work is part of the TransCampus project between TU Dresden and King’s College London and was partially supported by the U.S. Department of Veterans Affairs (5101CX001219) and the U.S. Department of Defense (W81XWH-16-1-0773)

EFFECT OF TUMOR NECROSIS FACTOR DEFICIENCY ON BEHAVIOR AND METABOLISM OF BIOGENIC AMINES IN BRAIN

In this study, we investigated the effect of knockout of tumor necrosis factor (TNF) gene on behavior and metabolism of biogenic amines in the brain. It was shown that knockout mice spend less time in Morris water maze in the sector where the platform was previously during probe trial. In the “novel object test” knockout mice sniff “object” less time than wild type mice. Furthermore, it was shown that the levels of 5-HIAA in the midbrain, the hippocampus and olfactory bulb significantly higher in knockout mice than in wild type mice