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- Materials and methods
Glycoprotein gp130 is involved in the interleukin-6 (IL-6) and related cytokines' signaling. Linkage between the gp130 coding gene and freezing reaction (catalepsy) was shown. Here, we compared the expression and function of the gp130 in male mice of catalepsy-resistant AKR/J strain and catalepsy-prone congenic AKR.CBA-D13Mit76 strain created by transferring the gp130 gene allele from catalepsy-prone CBA/Lac to the genome of AKR/J strain. No difference in the gp130 expression in the frontal cortex, hippocampus and midbrain between AKR and AKR.CBA-D13Mit76 mice was found. However, AKR.CBA-D13Mit76 mice were more sensitive to bacterial lipopolysaccharide (LPS). The administration of LPS (50 µg/kg, ip) significantly increased mRNA level of the gene coding IL-6-regulated glial fibrillary acidic protein (GFAP) in the midbrain, induced catalepsy and decreased locomotion in the open field and social investigation tests in AKR.CBA-D13Mit76, but not in AKR mice. The result indicates (1) the association between gp130 and hereditary catalepsy, (2) increased functional activity rather than expression of gp130 in AKR.CBA-D13Mit76 mice and (3) the involvement of gp130 in the mechanism of LPS-induced alteration of behavior.
Catalepsy (animal hypnosis, tonic immobility) is a state of prolonged motor inhibition characterized by plastic muscular tonus and failure to correct an externally imposed, awkward posture. The phenomenon represents a kind of passive defensive behavior and is found in vertebrates (Dixon 1998; Klemm 1989). An exaggerated form of catalepsy is a syndrome of some grave mental disorders such as schizophrenia and malignant neuroleptic syndrome (Singerman & Raheja 1994; Taylor & Fink 2003). A pronounced catalepsy was shown in 54% of mice of CBA/Lac strain, while catalepsy was not detected in AKR/J mouse strain (Kulikov et al. 1993). Using quantitative trait loci (QTL) analysis (Kulikov et al. 2003), selective breeding (Kondaurova et al. 2006) and genetic recombination (Kulikov et al. 2008a), the main gene of catalepsy was mapped on the 61–70 cM fragment of mouse chromosome 13. This fragment contains four genes expressed in the brain and, therefore, could be considered as putative candidate genes for catalepsy: Map3k1 (MAP kinase kinase kinase 1), Il6st (gp130 signal transducer), Gzmk (granzyme K) and Hspb3 (heat-shock protein 3) (Kulikov et al. 2008a).
Among them, the Il6st gene coding the glycoprotein gp130 attracts special attention. This glycoprotein is the signal transducer subunit in the receptors of so-called gp130-related cytokines such as interleukin (IL)-6, IL-11 and IL-27, ciliary neurotrophic factor, leukemia inhibitory factor, oncostatin M, cardiotrophin-1, cardiotrophin-like cytokine and neurotrophin (Chesnokova & Melmed 2002; Fasnacht & Muller 2008; Heinrich et al. 2003). The gp130-related cytokines are implicated in the regulation of immunity, neuronal plasticity, neurogenesis, inflammation as well as in the mechanisms of sickness behavior and mental disorders (Bluthe et al. 2000; Hayley et al. 2005; Naka et al. 2002). Endotoxin of outer membrane of gram-negative bacteria lipopolysaccharide (LPS) increased IL-6 secretion, attenuated locomotion, sensitivity to reward and grooming, induced anorexia, depression (Dantzer 2001) and catalepsy (Bazovkina & Kulikov 2009).
It was hypothesized that high predisposition to catalepsy observed in mice of CBA/Lac strain was produced by an alteration of the Il6st gene expression and/or the gp130 functional activity. To test this hypothesis, we used the AKR.CBA-D13Mit76 congenic mouse strain created by transferring the CBA allele of the Il6st gene to the genome of catalepsy-resistant AKR/J strain. AKR.CBA-D13Mit76 mice showed CBA-like catalepsy expression (50%) (Kulikov et al. 2008a) and were more aggressive than mice of the parental strains (Kondaurova et al. 2010). The differences between AKR.CBA-D13Mit76 and AKR/J mice in the Il6st gene expression and/or in the effects of LPS on locomotion and grooming in the open field test and social contacts (including aggression) in the social investigation test were expected.
The main aim of the study was to compare the Il6st gene expression and the gp130 sensitivity to LPS in AKR and AKR.CBA-D13Mit76 mice. It was intended to study the effects of LPS on (1) catalepsy, (2) behavior in the open field and social investigation tests and (3) expression of gp130-regulated glial fibrillary acidic protein (GFAP) coding gene in AKR and AKR.CBA-D13Mit76 mice.
Materials and methods
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- Materials and methods
The experiments were carried out on adult mouse males (8 weeks old, weighting 25 ± 2 g) of AKR/J (n = 43) and congenic AKR.CBAD13Mit76 (n = 48) strains. The AKR/J strain was maintained in the Institute of Cytology and Genetics by brother–sister inbreeding for at least 50 generations and was highly inbred. The AKR.CBAD13Mit76 strain was created in the Institute of Cytology and Genetics by transferring the 55–70 cM fragment [marked with microsatellites D13Mit74 (59 cM), D13Mit76 (61 cM) and D13Mit214 (71 cM)] of chromosome 13 (Fig. 1) from CBA/Lac strain to the genome of AKR/J strain using nine successive backcrossings to AKR/J strain. The backcrosses heterozygous for the CBA alleles of D13Mit74 and D13Mit76 markers and homozygous for the AKR allele of D13Mit214 marker were crossed with AKR mice. This genetic procedure washed out CBA alleles of all genes except those located in the controlled fragment. As a result the AKR.CBAD13Mit76 mice have the CBA allele of Il6st gene on the AKR genetic background (Kulikov et al. 2008a). The AKR.CBAD13Mit76 strain was maintained in the Institute of Cytology and Genetics by brother–sister inbreeding for at least 15 generations.
Figure 1. Structure of the 55–70 cM fragment of chromosome 13 transferred from CBA/Lac strain to the genome of AKR/J strain. The polymorphic microsatellite markers and Il6st gene positions are shown.
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After weaning, mice were separated by sex and kept as six per cage (40 × 30 × 15 cm) until age of 8 weeks under standard conditions (temperature: 18–22°C, relative humidity: 50–60%, standard food and water ad libitum). Two days before the experiment, the animals were isolated in cages of the same size to eliminate group effect. All experimental procedures were in compliance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). All efforts were made to minimize the number of animals used and their suffering.
Lipopolysaccharide (Escherichia coli 055:B5; Sigma-Aldrich Inc., St. Louis, MO, USA) was diluted in saline and injected intraperitoneally in the doses of 50 and 200 µg/kg 3 h prior to behavioral tests.
The effect of LPS (50 µg/kg) on catalepsy was tested in 10 AKR and 11 AKR.CBA-D13Mit76 mice. The control animals (9 AKR and 10 AKR.CBA-D13Mit76) were injected with saline.
Catalepsy was tested according to early described and verified procedure (Kulikov et al. 1993). Animals were firmly pinched between two fingers for 5 seconds at the scruff of the neck, placed on parallel bars, with the forepaws at 5 cm above the hind legs and then were released gently. The catalepsy duration was timed from the instant the animals were released to the instant the animals shifted their front paws from their initial position on the upper bar or made gross body or head movements. A trial ended either when an animal started to move or after 120 seconds of freezing. Immobility time of more than 20 seconds was considered as positive (cataleptic) response. Every animal was successively tested with 2-min intervals (the mouse was placed in its home cages between the trials) until three positive responses were achieved, but no more than 10 times. A mouse displaying three positive responses was scored as cataleptic, as only real cataleptic was able to reproduce its immobility 3 times. Immobility time was calculated as the mean of three trials with the maximal values.
The effects of LPS (50 and 200 µg/kg) on behavior in the social investigation and open field tests were studied in 15 AKR (n = 7 for 50 µg/kg and n = 8 for 200 µg/kg) and 17 AKR.CBA-D13Mit76 (n = 8 for 50 µg/kg and n = 9 for 200 µg/kg) mice. The control animals (9 AKR and 10 AKR.CBA-D13Mit76) were injected with saline.
In the social investigation test, in the home cage of tested male (resident) a young (4 weeks old) male (intruder) of white random-bred mice was placed and the resident behavior toward the intruder was recorded for 10 min with a digital camera (Sony, Japan). The total number of contacts (sniffing, grooming and attack) of the resident toward the intruder was counted as a measure of social interest. The percentage of residents which attacked intruders was used as a measure of aggression.
The open field test was started 10 min after the social investigation test and it was carried out on a circle arena (40 cm in diameter) bordered with white plastic wall (25 cm high) and illuminated through the mat and semitransparent floor with two halogen lamps of 12 W each placed 40 cm under the floor. This inverted illumination provided the maximal contrast between white animal and the arena (Kulikov et al. 2008b). Mice were placed near the wall and its movements were tracked for 5 min with a digital camera (Sony) placed 80 cm above the arena. The horizontal locomotor activity (distance traveled, cm), time in the center (20 cm in diameter) (% of the total time), vertical activity (number of rears) and ambivalent behavior (time of grooming) were calculated.
The behavioral characteristics in the social investigation and open field tests were evaluated using the EthoStudio software (Version 2) (Kulikov et al. 2008b).
An hour after the open field test, the animals treated with saline (control) and with 50 µg/kg LPS were decapitated, their cortexes, hippocampuses and midbrains were dissected, frozen with liquid nitrogen and kept at −70°C until RNA extraction. Total RNA was extracted with guanidine thiocyanate-phenol-chloroform mixture (Chomczynski & Sacchi 1987), treated with RNA-free DNAse (Promega, USA) and diluted with water treated with diethyl pyrocarbonate to 0.125 µg/µl. The samples of total RNA were tested for genomic DNA contamination using polymerase chain reaction (PCR) specific for β-actin gene primers (Table 1). A 8-µl aliquot (1 µg) of the total RNA was taken for cDNA synthesis with a random hexanucleotide mixture (Kulikov et al. 2005; Naumenko & Kulikov 2006; Naumenko et al. 2008). The genomic DNA contamination in the cDNA samples was tested using PCR specific for mouse tryptophan hydroxylase 1 gene primers (Table 1) (Kulikov & Naumenko 2007; Naumenko & Kulikov 2006). No trace of genomic DNA in the cDNA samples was detected at 36 cycles. The number of RNA polymerase II, gp130 and GFAP coding copies in the cDNA was evaluated with quantitative PCR using selective primers (Table 1) and mouse genomic DNA as external standard (200 copies/ng of genomic DNA). The expression of genes coding gp130 and GFAP was evaluated as the number of the copies with respect to 100 copies of RNA polymerase II gene (Kulikov et al. 2005; Kulikov & Naumenko 2007; Naumenko et al. 2008).
Table 1. Sequences and annealing temperatures of primers used for quantitative RT-PCR
|Gene||Sequence||Annealing temperature (°C)||PCR product size (bp)|
| ||R: 5′-acccacactgtgcccatcta-3′|| || |
|Tryptophan hydroxylase 1||F: 5′-gcttcaaagacaatgtctatcgtagaag-3′||60||164|
| ||R: 5′-ggcgtgggtcgggtagagtttgttt-3′|| || |
|RNA polymerase II||F: 5′-gttgtcgggcagcagaatgtag-3′||63||188|
| ||R: 5′-tcaatgagaccttctcgtcctcc-3′|| || |
|Il6st (gp130)||F: 5′-atttgtgtgctgaaggaggc-3′||63||186|
| ||R: 5′-aaaggacaggatgttgca-3′|| || |
| ||R: 5′-gcaaagttgtccctctccac-3′|| || |
The percentages of cataleptic and aggressive mice were compared with the Fisher's exact test. The vertical and horizontal locomotor activities, time in the center, time of grooming, time of catalepsy, number of social contacts and mRNA level were presented as means ± SEM and compared with two-way analysis of variance (anova) followed by the Fisher's post hoc analysis.
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Catalepsy was shown in 1 out of 10 saline-treated AKR mice and in 9 out of 10 control AKR.CBAD13Mit76 animals (P < 0.001). In the LPS-treated (50 µg/kg) group of AKR mice, 1 out of 10 was found cataleptic, while all 11 LPS-treated AKR.CBAD13Mit76 mice were cataleptics.
Time of catalepsy in the saline-treated AKR.CBAD13Mit76 mice was significantly higher compared with the control AKR mice (F1,36 = 135.7, P < 0.001; Fig. 2). Moreover, the time of catalepsy in the control AKR mice did not differ from zero (4.3 ± 7.6 seconds). Significant effects of LPS (F1,36 = 8.6, P < 0.006) and genotype × LPS interaction (F1,36 = 5.3, P < 0.032) on the catalepsy time were shown. Lipopolysaccharide (50 µg/kg) did not affect the time of catalepsy in AKR, but significantly increased it in AKR.CBAD13Mit76 mice (from 72 ± 7.2 seconds in saline to 110 ± 6.9 seconds in LPS treated, P < 0.0005; Fig. 2).
Figure 2. Time of cataleptic freezing (seconds) in the saline- (white bars) and LPS-treated (50 μg/kg) (gray bars) AKR and AKR.CBA-D13Mit76 mice. The data are means of three trials with the maximal values of freezing. ***P < 0.001 compared with saline-treated AKR.CBA-D13Mit76 mice, ##P < 0.01 compared with saline-treated AKR mice.
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In the open field test, the saline-treated mice of AKR and AKR.CBAD13Mit76 strains did not differ in the distance traveled (F1,45 = 2.2, P > 0.05; Fig. 3a), number of rears (F1,45 < 1, P > 0.05; Fig. 3b) and time in the center (F1,45 = 2.5, P > 0.05; Fig. 3c). However, the control mice of AKR.CBAD13Mit76 strain exceeded the control animals of AKR strain in the time of grooming (P < 0.01; Fig. 3d). Lipopolysaccharide decreased dose dependently the distance traveled (F2,45 = 6.1, P < 0.005; Fig. 3a), number of rears (F2,45 = 7.2, P < 0.002; Fig. 3b) and the grooming time (F2,45 = 7.8, P < 0.001; Fig. 3d) in mice of both strains. Significant genotype × LPS interaction was shown only for the time of grooming (F2,45 = 8.8, P < 0.001), while the interaction for the distance traveled, time in the center and number of rears was not significant (F2,45 < 1, P > 0.05) because LPS in the dose of 200 µg/kg decreased these behavioral parameters down to similar values in mice of AKR and AKR.CBAD13Mit76 strains. At the same time, the sensitivity of these behavioral parameters to LPS was different in AKR and AKR.CBAD13Mit76 mice. In AKR mice, significant attenuation of the distance traveled (P < 0.02), number of rears (P < 0.02) and grooming time (P < 0.03) was shown only at high dose of LPS (200 µg/kg), while lower dose of LPS (50 µg/kg) was ineffective. At the same time, in AKR.CBAD13Mit76 mice even low LPS dose (50 µg/kg) significantly decreased the distance traveled (P < 0.03), number of rears (P < 0.02) and grooming time (P < 0.001). No effect of LPS on the time in the center was detected (F2,45 < 1, P > 0.05; Fig. 3c).
Figure 3. Distance traveled (a), number of rears (b), time in the center (c) and grooming duration (d) in the open field test in AKR and AKR.CBA-D13Mit76 mice treated with saline (white bars), 50 μg/kg (gray bars) and 200 μg/kg (dashed bars) of LPS.*P < 0.05,**P < 0.01,***P < 0.001 compared with corresponding saline-treated control;##P < 0.01 compared with saline-treated AKR.
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The saline-treated AKR.CBAD13Mit76 mice investigated intruder more frequently compared with the control AKR mice (33.5 ± 2.9 in AKR.CBAD13Mit76 and 21.0 ± 0.3 in AKR, P < 0.005). The significant effect of LPS (F2,45 = 31.8, P < 0.001) and genotype × LPS interaction (F2,45 = 7.5, P < 0.002; Fig. 4a) was shown. In AKR mice, a considerable decrease in the number of contacts was shown only at the high dose of LPS (P < 0.003), while in AKR.CBAD13Mit76 mice both doses of 50 µg/kg (P < 0.001) and 200 µg/kg (P < 0.001) of LPS decreased the number of contacts (Fig. 4a).
Figure 4. Total number of contacts with juvenile intruder (a) and percentage of aggressive mice (b) in the social investigation test in AKR and AKR.CBA-D13Mit76 mice treated with saline (white bars), 50 μg/kg (gray bars) and 200 μg/kg (dashed bars) of LPS.*P < 0.05,**P < 0.01,***P < 0.001 compared with corresponding saline-treated control;#P < 0.05,##P < 0.01 compared with saline-treated AKR.
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A significant difference between the saline-treated mice of AKR and AKR.CBAD13Mit76 strains in the aggression toward young male was shown (Fig. 4b): 2 out of 9 (22%) AKR and 7 out of 10 (70%) AKR.CBAD13Mit76 saline-treated males attacked young intruders (P < 0.05). Both doses of LPS did not affect the number of aggressive males in AKR strain (2 out of 7 after 50 µg/kg and 1 out of 8 after 200 µg/kg of LPS). At the same time, LPS significantly decreased the number of aggressive males in AKR.CBA-D13Mit76 strain (down to 1 out of 8, P < 0.02 after 50 µg/kg and 1 out of 9, P < 0.015 after 200 µg/kg of LPS) (Fig. 4b).
The saline-treated mice of AKR and AKR.CBA-D13Mit76 strains did not differ in the Il6st mRNA levels in the cortex (F1,20 < 1, P > 0.05), hippocampus (F1,25 = 1.4, P > 0.05) and midbrain (F1,26 < 1, P > 0.05) (Fig. 5a). Lipopolysaccharide (50 µg/kg) did not affect the Il6st gene expression in the cortex (F1,20 < 1, P > 0.05), hippocampus (F1,25 < 1, P > 0.05) and midbrain (F1,26 < 1, P > 0.05) (Fig. 5a).
Figure 5. Expression of gp130 (a) and GFAP (b) genes in the cortex, hippocampus and midbrain in AKR and AKR.CBA-D13Mit76 mice treated with saline (white bars) and 50 μg/kg (gray bars) of LPS.**P < 0.01 compared with corresponding saline-treated control;#P < 0.05,##P < 0.01 compared with saline-treated AKR.
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No difference in the GFAP mRNA level in the hippocampus of the saline-treated AKR and AKR.CBA-D13Mit76 mice was shown (F1,25 = 1.2, P > 0.05). However, the GFAP gene expression was significantly higher in the cortex (F1,16 = 7.8, P < 0.013) and midbrain (F1,26 = 6.3, P < 0.02) of the saline-treated AKR compared with those of the control AKR.CBA-D13Mit76 mice (Fig. 5b). Lipopolysaccharide did not affect the gene expression in the cortex (F1,16 < 1, P > 0.05) and hippocampus (P > 0.05) of AKR and AKR.CBA-D13Mit76 mice. No effect of LPS on the GFAP mRNA level in the midbrain of AKR mice (P > 0.05) was found. At the same time, LPS significantly increased the GFAP mRNA level in the midbrain of AKR.CBA-D13Mit76 mice (P < 0.01, Fig. 5b).
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Earlier, the linkage between the main gene of catalepsy and the Il6st gene was shown using QTL analysis (Kulikov et al. 2003) and selective breeding (Kondaurova et al. 2006). Moreover, the CBA allele of Il6st gene transferred to the genome of catalepsy-resistant AKR strain increased predisposition to catalepsy in AKR.CBA-D13Mit76 congenic strain up to the level of 50% observed in catalepsy-prone CBA/Lac strain (Kulikov et al. 2008a). It was hypothesized that CBA allele of the gene predisposed mice to catalepsy by means of alteration of the expression and/or function of gp130. AKR.CBA-D13Mit76 strain with the AKR genetic ground and the CBA-derived gp130 provides good possibility to compare the effects of the CBA- and AKR-derived allele of Il6st gene on the expression and functional activity of gp130.
In the present study, we found 90% of cataleptics in AKR.CBA-D13Mit76 strain (9 out of 10). This result confirms that AKR.CBA-D13Mit76 is indeed a catalepsy-prone strain. In addition to high risk of catalepsy, increased ambivalent behavior (grooming duration) and social interactions in the saline-treated AKR.CBA-D13Mit76 mice compared with those in the control AKR mice was found. Seventy percent of AKR.CBA-D13Mit76 males attacked and bit juvenile male placed into their home cages. This result agreed with our earlier published data that mice of AKR.CBA-D13Mit76 strain were more aggressive than animals of the parental AKR and CBA strains (Kondaurova et al. 2010). These increases of catalepsy, grooming, social interest and aggression seemed to result from the interaction of the CBA allele of Il6st gene with the AKR genetic ground.
The most important result of the present study was the association of the CBA allele of Il6st gene with elevated sensitivity to LPS. Indeed, the low (50 µg/kg) dose of LPS did not affect the time of cataleptic immobility, locomotor activity, grooming, social investigation and aggression in AKR mice, but dramatically altered these parameters in AKR.CBA-D13Mit76 mice with the CBA allele of Il6st gene transferred to the AKR genome. Earlier, we showed that high dose (200 µg/kg ) of LPS induced catalepsy in 50% of mice of catalepsy-resistant C57BL/6 and DBA2 strains, while lower dose (50 µg/kg) of LPS was not effective (Bazovkina & Kulikov 2009). Here, we also detected no effect of 50 µg/kg of LPS on immobility time in mice of catalepsy-resistant AKR strain. At the same time, this dose of LPS increased immobility time in mice of catalepsy-prone AKR.CBA-D13Mit76 strain up to the maximal value (120 seconds) determined by the test (see Materials and Methods). This result indicates the association of CBA-derived gp130 with hereditary catalepsy in mice.
Lipopolysaccharide affects behavior by stimulation of IL-1β, IL-6 and tumor necrosis factor-α (TNF-α) secretion (Dantzer 2001). These cytokines activate three different downstream molecular pathways. AKR and AKR.CBA-D13Mit76 mice share the AKR-derived mechanisms associated with IL-1β and TNF-α as well as the IL-6Rα subunit, janus kinases and STAT proteins associated with IL-6 signaling. These strains differ only in the CBA-derived gp130. As no difference between AKR and AKR.CBA-D13Mit76 mice in the Il6st mRNA level was found, the increased sensitivity to LPS in AKR.CBA-D13Mit76 mice seemed to result from some alterations in the gp130 functional activity. At the same time, similar behavior attenuation in AKR and AKR.CBA-D13Mit76 mice treated with the high dose of LPS is probably mediated though the mechanism associated with IL-1β, which is shared in these strains.
In this study, we found that LPS administration produced activation of GFAP expression in the midbrain in mice of AKR.CBA-D13Mit76 strain, but not in animals of AKR strain. Of course, the GFAP gene can be regulated by multiple signaling mechanisms, but all possible mechanisms, except the gp130, are similar in AKR and AKR.CBA-D13Mit76 strains. Therefore, this difference in the effects of LPS on the GFAP gene expression in AKR and AKR.CBA-D13Mit76 mice reflects the genetically defined distinctions of the gp130 in these strains.
It was suggested that LPS-induced behavioral alterations in AKR.CBA-D13Mit76 mice resulted from activation of IL-6 receptors in the midbrain. This conclusion is not unexpected as (1) IL-6 and LPS affected the brain serotonergic system (Dunn 2006); (2) cell bodies of serotonergic neurons are located in the midbrain; (Dahlstrom & Fuxe 1964; Jacobs & Azmitia 1992) and (3) serotonin is involved in the regulation of locomotion, catalepsy (Popova & Kulikov 1995), depressive-like behavior (Lesch 2004; Lucki 1998) and aggression (Popova 2006).
In the present study, we found that the low dose of LPS suppressed behavior in AKR.CBA-D13Mit76, but not AKR mice. As Il6st is the only gene of the whole LPS-mediating network that is different between the AKR and AKR.CBA-D13Mit76 strains, these results can be considered as an experimental evidence of involvement of gp130 and IL-6 in the mechanism of LPS-induced sickness and depressive-like behavior.
Thus, the results provide experimental evidence for an association between gp130 and hereditary catalepsy in mice. The CBA allele of Il6st gene affects the gp130 functional activity, increases sensitivity to LPS and ensures high predisposition to catalepsy in AKR.CBA-D13Mit76 mice. The AKR.CBA-D13Mit76 congenic strain provides a new experimental tool to study the role of gp130-related cytokines in the regulation of normal and pathological behavior.