Sirli Raud

Behavioural differences between C57BL/6 and 129S6/SvEv strains are reinforced by environmental enrichment

Housing in enriched environment has been advocated as a means for controlled variation of environmental conditions in transgenic studies to explore interactions between genes and surroundings. In the present study, behavioural phenotypes of C57Bl/6 (B6) and 129S6/SvEv (129) mice, housed in either standard laboratory conditions or environmentally enriched conditions, were explored. Housing in enriched conditions increased exploratory activity in the plus-maze and reduced habituation in the locomotor activity test in B6 mice, whereas in 129 mice increased hot plate latencies and reduced aggression were observed. Compared to B6, 129 strain displayed lower exploratory activity in the plus-maze and locomotor activity test, longer hot plate latencies, spent more time immobile in the forced swim test and engaged more in social interaction. These behavioural differences between the two strains were reproducible independent of pre-experimental housing conditions. Moreover, environmental enrichment accentuated dissimilarities between the strains in the plus-maze, locomotor activity, hot plate and forced swim test. By contrast, strain differences in anxiety-like behaviours in the plus-maze test and in aggressive encounters in the resident-intruder test were not reproducible in mice housed in alternative environmental conditions, suggesting a strong contribution of environmental factors to the development of these phenotypes. It is concluded that the application of environmental enrichment in addition to standard housing conditions is a meaningful approach for testing reproducibility of behavioural findings within one laboratory.

Wfs1-deficient mice display impaired behavioural adaptation in stressful environment

Wfs1-deficient mice were generated by disrupting the 8th exon of Wfs1 gene. Reproduction rates of homozygous Wfs1-deficient mice were slightly below the expected values, they displayed intolerance to glucose and overall lower body weight. The present behavioural study was performed in female Wfs1-deficient mice due to their milder metabolic disturbances. Non-fasting blood glucose levels did not differ between homozygous Wfs1-deficient mice and wild-type littermates. While there was no difference in baseline plasma corticosterone, exposure to stress induced a nearly three-fold elevation of corticosterone in Wfs1-deficient mice in relation to wild-type littermates. Wfs1-deficient mice did not display obvious shortcomings in sensory and motor functioning as exemplified by intact responses in conditioned learning paradigms and rota-rod test. Locomotor activity of Wfs1-deficient mice was significantly lower only in brightly lit environment. Short-term isolation had a significant anxiogenic-like effect on the behaviour of Wfs1-deficient mice in dark/light exploration test. Lower exploratory activity of Wfs1-deficient mice in the plus-maze was antagonised by pre-treatment with diazepam (1 mg/kg), a GABA(A) receptor agonist. Wfs1-deficient mice displayed increased anxiety-like behaviour in hyponeophagia test. The locomotor stimulatory effects of amphetamine (2.5-7.5 mg/kg) and apomorphine (3 mg/kg) were significantly attenuated and facilitated, respectively, in Wfs1-deficient mice. There were no differences between Wfs1-deficient mice and wild-types in forced swimming behaviour and conditioned fear responses. Subtle impairments in reversal learning were apparent in Wfs1-deficient mice in the Morris water maze. Altogether, the present study demonstrates impaired behavioural adaptation of Wfs1-deficient mice in stress-inducing situations. It is likely that Wfs1 protein plays a major role in the behavioural adaptation mechanisms to novel and stressful environments.

Targeted mutation of CCK2 receptor gene antagonises behavioural changes induced by social isolation in female, but not in male mice

Neuropeptide cholecystokinin (CCK) regulates the adaptation of rodents in the novel environment. In the present study we analysed the behavioural changes induced by the individual housing in mice, lacking CCK(2) receptors. The wild-type (+/+) and homozygous (-/-) CCK(2) receptor deficient mice of both gender were used throughout the study. The weight gain during the 21-day isolation period and changes in the locomotor activity following the social separation were measured. The elevated plus-maze and resident/intruder tests were also performed to test alterations in the emotional behaviour. Social isolation induced locomotor hyperactivity, reduced weight gain and increased aggressiveness in the wild-type (+/+) and homozygous (-/-) male mice. In the wild-type (+/+) female mice the significant reduction of exploratory activity in the plus-maze was evident. By contrast, in female mice, lacking CCK(2) receptors, the exploration of the plus-maze was not significantly affected by the individual housing. This finding demonstrates that the social isolation does not cause anxiety-like state in the CCK(2) receptor deficient mice. Moreover, the targeted invalidation of CCK(2) receptors increased in male mice the affinity of dopamine D(2) receptors in the sub-cortical structures, whereas in female mice the increased affinity of 5-hydroxytryptamine(2) (5-HT(2)) receptors in the frontal cortex was established. The increased affinity of 5-HT(2) receptors is probably the compensatory change to the lack of CCK(2) receptors in female mice and probably reflects the reduced sensitivity of these animals to the anxiogenic manipulations. In conclusion, targeted mutation of CCK(2) receptors selectively antagonised the behavioural changes induced by the individual housing in females, but not in male mice.

Relation between increased anxiety and reduced expression of alpha1 and alpha2 subunits of GABAA receptors in Wfs1-deficient mice

Mutations in the coding region of the WFS1 gene cause Wolfram syndrome, a rare multisystem neurodegenerative disorder of autosomal recessive inheritance. In clinical studies a relation between mutations in the Wfs1 gene and increased susceptibility for mood disorders has been established. According to our previous studies, mice lacking Wfs1 gene displayed increased anxiety in stressful environment. As the GABA-ergic system plays a significant role in the regulation of anxiety, we analyzed the expression of GABA-related genes in the forebrain structures of wild-type and Wfs1-deficient mice. Experimentally naïve Wfs1-deficient animals displayed a significant down-regulation of alpha1 (Gabra1) and alpha2 (Gabra2) subunits of GABA(A) receptors in the temporal lobe and frontal cortex. Exposure of wild-type mice to the elevated plus-maze decreased levels of Gabra1 and Gabra2 genes in the temporal lobe. A similar tendency was also established in the frontal cortex of wild-type animals exposed to behavioral test. In Wfs1-deficient mice the elevated plus-maze exposure did not induce further changes in the expression of Gabra1 and Gabra2 genes. By contrast, the expression of Gad1 and Gad2 genes, enzymes responsible for the synthesis of GABA, was not significantly affected by the exposure of mice to the elevated plus-maze or by the invalidation of Wfs1 gene. Altogether, the present study demonstrates that increased anxiety of Wfs1-deficient mice is probably linked to reduced expression of Gabra1 and Gabra2 genes in the frontal cortex and temporal lobe.

Heterozygous mice with Ric-8 mutation exhibit impaired spatial memory and decreased anxiety.

Ric-8 is a guanine nucleotide exchange factor for a subset of Galpha proteins and it is required to maintain Galpha(q) and the Galpha(s) pathways in functional state. In adult mice Ric-8 is expressed in regions involved in the regulation of behavior (neocortex, cingulate cortex and hippocampus). As Ric-8 is shown to regulate neuronal transmitter release, the aim of present study was to perform behavioral analysis of ric-8 mutant. Homozygous (-/-) ric-8 mutant mice are not viable and die in early embryonic development, therefore for behavioral analysis heterozygous (+/-) ric-8 mutant mice were used. We found decreased anxiety of ric-8 heterozygous mice in light-dark compartment test where mutant mice significantly avoided the light compartment. In spatial learning paradigm (Morris water maze) the performance of ric-8 (+/-) mice was impaired. Namely, in the reversal test, ric-8 (+/-) mice exhibited significant delay to find the hidden platform compared to wild-type (wt) littermates. We did not find differences in the behavioral tests reflecting the motor abilities of mice (motor activity, rota-rod). Therefore, described alterations seem to be specific for anxiety and spatial learning. Based on these results we can conclude the importance of ric-8 in the regulation of memory and emotional behavior.

Lower anxiety and a decrease in agonistic behaviour in Lsamp-deficient mice.

In rodents, the Lsamp gene has been implicated in trait anxiety, fear reaction and fear conditioning. Human data link the LSAMP gene to several psychiatric disorders. In this study, we presented a general phenotypic characterization of Lsamp gene-deficient mouse line, created by deleting exon 1b. These mice displayed no gross sensory-motor deficiencies, no overt abnormalities and performed normally in memory and learning tests. However, they responded with increased activity to new environments. Moreover, they displayed reduced anxiety and notable deviations in social behaviour, such as lack of whisker trimming, reduced aggressiveness and reduced dominance. One possible explanation for the anxiolytic-like effect of the deletion of the Lsamp gene is a shift in balance in the Gabra1 and Gabra2 genes in the temporal lobe in favor of the Gabra2 transcript, encoding α2 subunit of GABA(A) receptors that mediate the stimulating effect of GABA agonists. The overall phenotype of Lsamp-deficient mice, characterized by decreased anxiety and several alterations in social behaviour, makes them a good model for studying the molecular mechanisms behind inadequate social behaviours observed in several psychiatric disorders.

Different housing conditions alter the behavioural phenotype of CCK2 receptor-deficient mice

The behavioural phenotype of mice, lacking CCK(2) receptors, has varied across studies conducted not only in different laboratories, but also across studies published by the same laboratory. The present study was designed to elucidate the phenotype of CCK(2) receptor-deficient mice housed in two different environmental conditions within the same laboratory. Environmental enrichment was used as an alternative environment to standard laboratory conditions. Significant genotype by environment interaction was observed in the plus-maze, hot-plate, restraint-induced analgesia and water maze test. While mice, lacking CCK(2) receptors, housed in standard conditions were more anxious, displayed stronger restraint-induced analgesia and performed worse in the water maze when compared to corresponding wild-type littermates, none of these phenotypes were observed in mice, housed in enriched conditions. By contrast, in the hot-plate test, rota-rod and locomotor activity test a genotype-dependent phenotype was observed in mice housed in enriched, but not in standard conditions. Moreover, the phenotype of CCK(2) receptor-deficient mice established in the hot-plate test and rota-rod was sex-specific. These results suggest that thorough and labour-consuming study of mutation-induced behavioural phenotype is necessary not only in different genetic backgrounds but also the substantial variation of phenotype due to sex- and environment-related factors have to be explored.

Distinct changes in the behavioural effects of morphine and naloxone in CCK2 receptor-deficient mice

The effects of morphine, mu-opioid receptor agonist, and naloxone, a non-selective opioid receptor antagonist, in the locomotor activity and place conditioning tests were studied in the CCK(2) receptor-deficient male mice. The exposure of mice to the motility boxes for 3 consecutive days induced a significant inhibition of locomotor activity in the wild-type (+/+) mice compared to homozygous (-/-) animals. The administration of naloxone (10 mg/kg i.p.) to animals, adapted to the motility boxes, induced a significant reduction of locomotor activity in the homozygous (-/-), but not in the wild-type (+/+) mice. Treatment of habituated mice with morphine (10 mg/kg i.p.) caused a stronger increase of locomotor activity in the wild-type (+/+) mice compared to the homozygous (-/-) littermates. In the place preference test the pairing of the preferred side with naloxone (1 and 10 mg/kg i.p.) induced a dose-dependent place aversion in the wild-type (+/+) mice. The treatment with naloxone was less effective in the homozygous (-/-) mice, because the high dose of naloxone (10 mg/kg) tended to shift the preference. The pairing of morphine (3 mg/kg i.p.) injections with the non-preferred side induced a significant place preference both in the wild-type (+/+) and homozygous (-/-) mice. The increased density of opioid receptors was established in the striatum of homozygous (-/-) mice, but not in the other forebrain structures. In conclusion, the targeted invalidation of CCK(2) receptors induces a dissociation of behavioural effects of morphine and naloxone. Morphine-induced place preference remained unchanged, whereas hyper-locomotion was less pronounced in the mutant mice compared to the wild-type (+/+) littermates. By contrast, naloxone-induced place aversion was weaker, but naloxone caused a stronger inhibition of locomotor activity in the homozygous (-/-) mice than in the wild-type (+/+) animals. These behavioural alterations can be explained in the light of data that the targeted mutation of CCK(2) receptors induces distinct changes in the properties of opioid receptors in various brain structures.

Deletion of the Lsamp gene lowers sensitivity to stressful environmental manipulations in mice.

The Lsamp gene gives rise to limbic system-associated membrane protein (LAMP), which is expressed on the surface of somata and proximal dendrites of neurons. Lsamp-deficient mice have been shown to be slightly hyperactive in novel environments and less anxious, and they display alterations in swimming speed, fear reaction, fear conditioning and social behaviour. In human studies, links between the LSAMP gene and several psychiatric disorders have been found and LSAMP has been established as a tumour suppressor gene. To study the impact of environmental manipulations on the phenotype, we exposed male Lsamp-deficient mice to environmental enrichment (EE), a technique that has often been shown to abolish phenotypic deviations in knockout mice, and to social isolation, a stressful manipulation, after which all the mice were tested in a behavioural battery. EE abolished differences between the genotypes in body weight and anogenital sniffing, a behaviour related to aggressiveness, and amplified the anxiolytic-like phenotype of Lsamp-deficient mice both in the plus maze and motility box. Isolation abolished differences between the genotypes in body weight and anxiety and amplified the differences in swimming speed and anogenital sniffing. EE and isolation failed to modify the results as compared to standard housing in whisker trimming, locomotor activity, marble burying and corticosterone levels. In conclusion, Lsamp-deficient mice were less sensitive to isolation stress than their wild-type littermates. Lack of LAMP protein seemingly leads to a deterioration in the ability to adapt to novel stressful environments and stimuli.

Cat odour exposure decreases exploratory activity and alters neuropeptide gene expression in CCK2 receptor deficient mice, but not in their wild-type littermates

An attempt was made to establish whether the anxiogenic effect of cat odour differs in female wild-type and CCK(2) receptor deficient mice, having different exploratory activity in the elevated plus-maze. The exposure of wild-type and homozygous CCK(2) receptor deficient mice to cat odour did not reveal substantial differences between the two genotypes. The number of contacts with the cat odour impregnated cloth was reduced and the frequency of stretch-attend postures was increased similarly in wild-type and homozygous mice. However, the following exposure of mice to the elevated plus-maze established differences as homozygous mice displayed increased exploratory activity in the plus-maze. The cat odour exposure significantly reduced exploratory activity only in homozygous mice. Together with the increased exploratory activity we established in homozygous mice significantly increased expression of the Oprm1 gene in the frontal cortex and mesencephalon. The exposure of mice to cat odour caused only minor changes in the gene expression of wild-type mice, whereas in homozygous animals a significantly increased expression of the Mc3r gene in the frontal cortex and temporal lobe, and the Pomc1 gene in the temporal lobe, mesencephalon and mesolimbic area was established. In conclusion, CCK(2) receptor deficient mice displayed reduced anxiety compared to their wild-type littermates in the plus-maze test. This behavioural effect seems to be related, at least partly, to an increased tone of opioid system in the brain. Moreover, homozygous mice respond to the exposure of cat odour with an increased anxiety. This effect seems to be related to the increased function of the melanocortin system in the brain structures of genetically modified mice.