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On the association between lipopolysaccharide induced catalepsy and serotonin metabolism in the brain of mice genetically different in the predisposition to catalepsy
Pharmacology, Biochemistry and Behavior 111 (2013) 71–75
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On the association between lipopolysaccharide induced catalepsy and serotonin metabolism in the brain of mice genetically different in the predisposition to catalepsy Ekaterina Yu. Bazhenova ⁎, Alexander V. Kulikov, Maria A. Tikhonova, Daria V. Bazovkina, Daria V. Fursenko, Nina K. Popova Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, Novosibirsk 630090, Russian Federation
a r t i c l e
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Article history: Received 21 June 2013 Received in revised form 6 August 2013 Accepted 10 August 2013 Available online 28 August 2013 Keywords: Lipopolysaccharide Serotonin metabolism Catalepsy Brain Mice
a b s t r a c t The study of the interaction between nervous and immune systems in the mechanism of psychopathology is an important problem of neuroscience. Catalepsy (freezing reaction) is a passive defensive strategy in response to threatening stimuli. An exaggerated form of catalepsy is a syndrome of some grave mental disorders. Both the brain serotonin (5-HT) and immune systems were shown to be involved in the mechanism of catalepsy. Here we compared the effects of two doses (50 or 200 μg/kg, ip) of innate immune system activator, bacterial lipopolysaccharide (LPS), on catalepsy, 5-HT and its main metabolite, 5-hydroxyindole acetic acid (5-HIAA) in the hippocampus, striatum, and midbrain of mice of catalepsy-prone (CBA/Lac and AKR.CBA-D13Mit76) and catalepsyresistant (AKR/J) strains. The expression of LPS-induced catalepsy as well as 5-HIAA/5-HT ratio in the midbrain and striatum were significantly higher in mice of the catalepsy-prone strains compared with animals of the catalepsy-resistant strains. These results indicated an involvement of the brain 5-HT system in the cataleptogenic effect of LPS and open up new vistas for understanding the nervous–immune mechanism of behavioral disorders. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Catalepsy (tonic immobility, immobility reflex, animal hypnosis) is a state of pronounced motor inhibition characterized by a failure to correct an externally imposed, awkward posture. In some wild animals it is an element of natural freezing response to predator appearance (Dixon, 1998). An exaggerated form of catalepsy is the syndrome of grave mental disorders such as schizophrenia, parkinsonism and extrapyramidal dysfunctions (Caroff et al., 2000; Daniels, 2009; Lee, 2007, 2010; Paparrigopoulos et al., 2009; Sanberg et al., 1988; Weder et al., 2008). Drug-free catalepsy can be induced by pinching mice at the scruff of the neck (Amir et al., 1981). This pinch-induced catalepsy is a very rare phenomenon that is not found in mice of the most common inbred strains, such as C57BL/6J, DBA/2, AKR/J, etc. But about 50% of CBA/Lac mice showed catalepsy after several pinches (Kulikov et al., 1993). The main locus for catalepsy was mapped to the 61–70-cM fragment of chromosome 13 (Kulikov et al., 2008). Then, the CBA-derived 59–70 cM fragment of chromosome 13 containing the main locus
⁎ Corresponding author at: Laboratory of Behavioral Neurogenomics, Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, Lavrentyeva Ave. 10/2, 630090 Novosibirsk, Russian Federation. Tel.: +7 383 3636187; fax: +7 383 3331278. E-mail address: [email protected] (E.Y. Bazhenova). 0091-3057/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pbb.2013.08.009
of catalepsy transferred to the AKR genome and the recombinant AKR.CBA-D13Mit76 (D13) strain was created. A pronounced catalepsy was found in about 50% of D13 mice (Kulikov et al., 2008). Earlier we showed the involvement of the brain serotonin (5-HT) and innate immune systems in the regulation of pinch-induced catalepsy in mice. The key enzyme of 5-HT synthesis, tryptophan hydroxylase, in the striatum of mice of catalepsy-prone CBA strain was increased compared with mice of catalepsy-resistant AKR and C57BL/6 strains (Kulikov et al., 1995). 5-HT1A receptor agonist 8-OH-DPAT attenuated hereditary catalepsy in mice (Kulikov et al., 2012). Bacterial lipopolysaccharide (LPS) or proinflammatory interleukin-6 (IL-6) induced catalepsy in mice of catalepsy-resistant C57BL/6 strain (Bazovkina et al., 2011). Moreover, the gene encoding gp130 protein, a subunit of IL-6 receptor, is linked to hereditary catalepsy (Kulikov et al., 2008). Although, LPS increases 5-HT metabolism in the brain of rats (Dunn, 2006), the effect of LPS on 5-HT metabolism in mice is contradictory. On one hand, LPS increased the level of the main 5-HT metabolite, 5-hydroxyindoleacetic acid (5-HIAA) in the brain of C3H/He mice (Dunn and Chuluyan, 1994). On the other hand LPS produced no effect on 5-HT metabolism in the brain of BALB/c mice (Cho et al., 1999). A possible genotype influence on the effect of LPS on the brain 5-HT system could be hypothesized. In order to test this hypothesis we studied the effect of LPS on the brain 5-HT metabolism and catalepsy in mice of catalepsy-resistant and catalepsy-prone strains.
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2. Methods 2.1. Animals and treatments The experiments were carried out on adult male mice (3– 4 months old, weighting 28 ± 4 g) of catalepsy-resistant AKR/J (n = 51), as well as catalepsy-prone CBA/Lac (n = 50) and recombinant AKR.CBAD13Mit76 (D13) (n = 51) strains. The D13 strain was created in the Institute of Cytology and Genetics by transferring the 59–70 cM fragment marked with microsatellites D13Mit74 (59 cM), D13Mit76 (61 cM) and D13Mit214 (71 cM) of chromosome 13 containing the major gene of catalepsy from CBA/Lac to the genome of AKR/J strain. D13 and AKR have the same genetic background and are distinguished by the CBA- or the AKR-derived 59–70 cM fragment, respectively. However, about 50% of D13 mice show CBA-like catalepsy, while none of AKR mice displays catalepsy (Kulikov et al., 2008). AKR, CBA and D13 strains were maintained at the Institute of Cytology and Genetics by brother–sister inbreeding for at least 55, 55 and 20 generations, respectively. After weaning, mice were separated by sex and kept six animals per cage (40 × 30 × 15 cm) until the age of 3–4 months 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 individually housed in cages of the same size to eliminate group effect. This two-day period of separation was shown to be sufficient to eliminate the group effect (Naumenko et al., 1971), but short to produce an isolation effect (Valzelli, 1973). 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. LPS (Escherichia coli 055:B5; Sigma-Aldrich Inc., St. Louis, MO, USA) was diluted in sterile saline and injected intraperitoneally in the doses of 50 or 200 μg/kg 3 h prior to behavioral test or decapitation. It was shown that the peak of sickness behavior was observed in 2–6 h after an ip LPS administration (Dantzer et al., 2008). These doses were chosen since earlier we showed that 200 μg/kg of LPS decreased locomotion and social behavior in D13 and AKR mice, while 50 μg/kg of LPS was effective only in D13 mice (Kulikov et al., 2010).
of 7 animals. Three hours after LPS or saline treatment the animals were decapitated and their striatum (caudate nucleus, putamen and globus pallidus), hippocampus (dentate gyrus, CA1 and CA3) and midbrain (n. raphe dorsalis and n. raphes medianus) were dissected, frozen with liquid nitrogen and kept at −70 °C until the measurement. Concentrations of 5-HT and 5-HIAA were assessed with high performance liquid chromatography (HPLC) (Kulikov et al., 2012). Tissue samples were homogenized in 200 μl of buffer containing 0.4 M HClO4 (Sigma, USA) and 0.27 mM EDTA (Ameresco, USA). The homogenates were centrifuged 5 min at 15000 g (4 °C) and supernatant was filtered through Whatman GF/C fiberglass filters (Whatman Int. Ltd., UK). Concentrations of 5-HT and 5-HIAA were then evaluated by HPLC on Nucleosil C8 column (Nucleosil C8 column, 3 μm particle size, L × I.D. 100 mm × 4.6 mm, Sigma-Aldrich, USA) with electrochemical detection (500 mV, Coulochem III, ESA, Inc., USA) and flowcell (BASInc, USA) using solvent delivery module LC-20AD (Shimadzu Corporation, Japan). The mobile phase contained NaH2PO4 (100 mM, pH = 4.5), 0.1 mM Na2EDTA, 1.4 mM 1-octanesulfonic acid sodium salt (Sigma, USA) and methanol (4 vol.%; Vekton Ltd., Russia). Its flow rate was 0.6 ml/min. Standard solution containing 2 ng of 5-HT and 5-HIAA was repeatedly assayed throughout the entire procedure. The concentration of 5-HT and 5-HIAA peaks was estimated by comparison of the magnitudes of corresponding picks with the respective external standards using the MultiChrom v.1.5 software (Ampersand Ltd., Russia) and expressed in μg per g of tissue sample. The intensity of 5-HT metabolism was evaluated by the 5-HIAA/5-HT ratio. The 5-HIAA/5-HT ratio reflects adequately the intensity of oxidative deamination of 5-HT and is more valid than the commonly used 5-HIAA level (Popova et al., 2001).
2.4. Statistics All values were presented as means ± SEM and compared with two-way analysis of variance (ANOVA) followed by the Fisher's post hoc analysis.
2.2. Test for catalepsy
3. Results
The effect of LPS (50 or 200 μg/kg) on catalepsy was tested in AKR (n = 10 for 50 μg/kg and n = 11 for 200 μg/kg), CBA (n = 8 for 50 μg/kg and n = 10 for 200 μg/kg) and D13 (n = 11 for 50 μg/kg and n = 9 for 200 μg/kg) mice. The control animals (9 AKR, 11 CBA and 10 D13) were injected with saline. Catalepsy was tested according to an earlier described procedure (Kulikov et al., 1993, 2008). Animals were pinched with two fingers for 5 s 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 s of freezing. Immobility time of more than 20 s was considered as positive (cataleptic) response. Every animal was successively tested with 2-min intervals (the mouse was placed in its home cage between the trials) until three positive responses were achieved, but no more than 10 times. Immobility time was calculated as the mean of three trials with the maximal values. The recovery of this procedure was about 80% (Kulikov et al., 1993).
3.1. Effect of LPS on catalepsy
2.3. Measurement of 5-HT metabolism The effects of LPS (50 or 200 μg/kg) on the concentration of 5-HT and its main catabolite — 5-hydroxyindoleacetic acid (5-HIAA) in the brain were studied in nine groups (three strains × three treatments)
Catalepsy was shown in 1 out of 10 saline-treated AKR mice, in 9 out of 10 control D13 animals and in 8 out of 11 control CBA mice. 50 μg/kg of LPS did not produce catalepsy in AKR mice (only 1 of 10 LPS-treated AKR showed catalepsy), but catalepsy was observed in all 11 D13 mice and 8 CBA mice treated with LPS. Eight out of 11 AKR as well as all D13 and CBA mice treated with 200 μg/kg showed catalepsy. Significant effects of genotype (F2,80 = 53.15, p b 0.001), LPS (F2,80 = 14.9, p b 0.001) and genotype × LPS interaction (F4,80 = 4.05, p b 0.01) on catalepsy duration were shown. Immobility time in the saline-treated D13 or CBA mice was higher than in AKR mice (p b 0.01). LPS at the dose of 50 μg/kg did not affect the time of catalepsy in AKR mice, but significantly increased it in D13 mice (from 72.03 ± 11.43 s in saline-treated to 115.9 ± 2.3 s in LPS-treated, p b 0.001; Fig. 1) and CBA mice (from 46.18 ± 8.7 s in saline-treated to 96.25 ± 9.91 s in LPS-treated, p b 0.001; Fig. 1). LPS at the dose of 200 μg/kg increased the time of catalepsy in all strains: AKR (p b 0.01), D13 (p b 0.05) and CBA (p b 0.001) (Fig. 1). No difference in catalepsy duration between CBA and D13 mice treated saline, 50 μg/kg or 200 μg/kg of LPS was revealed (p N 0.05). At the same time, immobility time in catalepsy-resistant AKR mice treated with saline (p b 0.01 vs CBA and p b 0.001 vs D13), 50 μg/kg (p b 0.0001) or 200 μg/kg (p b 0.01 vs CBA and p b 0.001 vs D13) of LPS was significantly lower compared with catalepsy-prone CBA or D13 mice.
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3.2. Effect of LPS on 5-HT metabolism
Fig. 1. Mean catalepsy duration (s) in the saline- and LPS-treated mice of catalepsy-resistant (AKR) and catalepsy-prone (CBA and D13) strains. All animals were ip injected with saline or LPS 3 h before the test. The numbers of mice: 1) AKR: saline — 9, 50 μg/kg of LPS — 10, 200 μg/kg of LPS — 11; 2) CBA: saline — 11, 50 μg/kg of LPS — 8, 200 μg/kg of LPS — 10; 3) D13: saline — 10, 50 μg/kg of LPS — 11, 200 μg/kg of LPS — 9. *p b 0.05, **p b 0.01, ***p b 0.001 vs corresponding saline-treated control; ##p b 0.01, ###p b 0.001 vs salinetreated AKR; &&&p b 0.001 vs LPS-treated (50 μg/kg) AKR; @@@p b 0.001 vs LPS-treated (200 μg/kg) AKR.
No effect of genotype on 5-HT level in the hippocampus (F2,54 b 1) and the midbrain (F2,54 b 1) was shown. At the same time, a significant effect of genotype on 5-HT level in the striatum was revealed (F2,54 = 3.5, p b 0.05): 5-HT concentration in this structure was higher in AKR mice compared with CBA (p = 0.052) and D13 (p b 0.01) mice. LPS decreased 5-HT level in the hippocampus (F2,54 = 7.6, p b 0.01) and striatum (F2,54 = 22.7, p b 0.001), but increased 5-HT concentration in the midbrain (F2,54 = 4.4, p b 0.05). LPS at the dose of 50 μg/kg significantly decreased 5-HT level in the hippocampus of AKR (p b 0.01) and D13 (p b 0.05) mice. At the same time, 200 μg/kg of LPS significantly decreased 5-HT level in the striatum of mice of the studied strains. LPS at the dose of 200 μg/kg (p b 0.05), but not 50 mg/kg increased 5-HT level in the midbrain of mice of CBA strain compared with the control mice. A tendency to increase was found in congenic D13 mice at both doses of LPS (p = 0.055 and p = 0.054) (Fig. 2A). Neither genotype (F2,54 = 2.0, p N 0.05), nor LPS (F2,54 = 3.1, p N 0.05) altered 5-HIAA level in the hippocampus. No difference in 5-HIAA concentration in the striatum (F2,54 b 1) or midbrain (F2,54 = 1.2, p N 0.05) of the saline-treated animals was found. At the same time, LPS significantly altered 5-HIAA level in these structures (F2,54 =
Fig. 2. 5-HT (A) and 5-HIAA (B) levels (μg/g of wet tissue mass) in the midbrain, hippocampus and striatum of the saline- and LPS-treated mice of catalepsy-resistant (AKR) and catalepsyprone (CBA and D13) strains. All animals were ip injected with saline or LPS 3 h before decapitation. The bars represent means ± SEM of seven values. *p b 0.05, **p b 0.01, ***p b 0.001 vs corresponding saline-treated control; #p b 0.05, ##p b 0.01 vs saline-treated AKR.
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3.6, p b 0.05 in the striatum and F2,54 = 11.3, p b 0.001 in the midbrain). While LPS at the dose of 200 μg/kg significantly decreased 5-HIAA in the striatum of AKR mice (p = 0.02), both doses of LPS significantly increased 5-HIAA level in the midbrain of CBA (p b 0.01 at 50 μg/kg and p b 0.001 at 200 μg/kg) and D13 (p b 0.01) mice (Fig. 2B). LPS increased 5-HT turnover (5-HIAA/5-HT rate) in the midbrain (F2,54 = 13.2, p b 0.001), hippocampus (F2,54 = 45.4, p b 0.001) and striatum (F2,54 = 12.2, p b 0.001). Both doses of LPS significantly increased 5-HT turnover in the hippocampus of mice of the three strains studied (p b 0.001). LPS at the dose of 50 μg/kg increased 5-HT turnover in the midbrain only of the catalepsy-prone CBA (p b 0.001) and D13 (p b 0.05) strains without any effect on this index in AKR mice. At the same time, 200 μg/kg of LPS significantly increased 5-HT turnover in this structure in all strains. Neither 50 μg/kg, nor 200 μg/kg of LPS affected 5-HT turnover in the striatum of AKR mice. However, LPS at the dose of 200 μg/kg significantly increased 5-HT metabolism in the striatum of catalepsy-prone CBA (p b 0.01) and D13 (p b 0.05) mice (Fig. 3). 4. Discussion Earlier we found that LPS (100 or 200 μg/kg) or IL-6 (7.5 or 10.0 μg/kg) induced catalepsy in mice of catalepsy-resistant C57BL/ 6 strain (Bazovkina et al., 2011). Here, we confirmed our earlier data and showed that 200 μg/kg of LPS produced catalepsy in mice of another catalepsy-resistant AKR strain. An important result of the present study was that LPS produced more pronounced cataleptogenic effect on mice of catalepsy-prone CBA and D13 strains than on mice of catalepsyresistant AKR strain. Although mice of AKR and D13 strains had the same AKR-derived genetic background (Kulikov et al., 2008), they differed in the cataleptogenic effect of the low dose of LPS (50 μg/kg). This dose failed to induce catalepsy in AKR mice, but significantly increased the time of cataleptic immobility in D13 and CBA mice. Since D13 and CBA mice have the same CBA-derived 59–70 cM fragment of chromosome 13 containing the main locus of catalepsy (Kulikov et al., 2008), this result indicated an association between the main locus of catalepsy and the cataleptogenic effect of LPS. An intriguing question concerns the mechanism of this association between hereditary catalepsy and the cataleptogenic effect of LPS. There are data that show that the brain 5-HT system is involved in the mechanism of catalepsy in rats. Agonists of 5-HT1A receptor dramatically reduced neuroleptic-induced (Wadenberg, 1996) and hereditary (Kulikov et al., 1994) catalepsy. Blockade of 5-HT synthesis with pCPA, an irreversible inhibitor of the key enzyme of 5-HT synthesis, tryptophan hydroxylase 2 (TPH2), significantly reduced both neuroleptic-
Fig. 3. 5-HIAA/5-HT ratio in the midbrain, hippocampus and striatum of the saline- and LPS-treated mice of catalepsy-resistant (AKR) and catalepsy-prone (CBA and D13) strains. All animals were ip injected with saline or LPS 3 h before decapitation. The bars represent means ± SEM of seven values. *p b 0.05, **p b 0.01, ***p b 0.001 vs corresponding salinetreated control.
induced (Kostowski et al., 1972) and hereditary catalepsy (Kulikov et al., 1992). Selective breeding for catalepsy increased TPH2 activity in the striatum of rats of Genetic Catalepsy (GC) strain (Kulikov et al., 1992). At the same time, the association between hereditary catalepsy and 5-HT system in mice is less clear. The only study showed a significant increase of TPH2 activity in the striatum of catalepsy-prone CBA mice compared to such catalepsy-resistant strains as AKR/J and C57BLl/6J (Kulikov et al., 1995). LPS increases blood levels of such proinflammatory cytokines as ILβ, IL-6, and TNFα (Dantzer, 2004). The data on the effects of LPS and IL-1β on 5-HT metabolism in the brain are rather contradictory. Majority of authors showed that LPS or IL-1β increased 5-HIAA concentration in rat brain (Dunn, 1992; Dunn and Wang, 1995; Dunn et al., 1999; Givalois et al., 1999; Hsieh et al., 2002; Lacosta et al., 1999; Lavicky and Dunn, 1995; Linthorst et al., 1995; Merali et al., 1997; Wang and Dunn, 1999). However, some authors did not find the effect of IL-1β on 5-HT metabolism (Kabiersch et al., 1988). In mice, the effect of LPS on 5-HT metabolism seems to depend on genotype: LPS increased 5HIAA level in the brain of C3H/HeJ and C3H/HeN mice (Dunn and Chuluyan, 1994), but not in BALB/c mice (Cho et al., 1999). At the present study, we found that in all studied strains LPS decreased 5-HT level in the hippocampus and striatum where the 5-HT endings are located and increased the level of 5-HT in the midbrain where the 5-HT bodies are located. At the same time, LPS significantly increased 5-HIAA level in the midbrain of mice of catalepsy-prone CBA and D13 strains only. LPS increased 5-HIAA/5-HT ratio in the hippocampus of all strains studied in a genotype-independent manner. At the same time, in the midbrain and striatum the effect of LPS on this trait depended on genotype: it was more prominent in mice of catalepsyprone strains than in animals of catalepsy-resistant strain. In a steadystate condition when all synthesized 5-HT was metabolized to 5-HIAA, an increase in 5-HIAA/5-HT ratio corresponds to activation of 5-HT metabolism. Therefore, the present results indicated that LPS produced more intensive effect on 5-HT metabolism in the midbrain and striatum of mice of genetically predisposed to catalepsy strains compared with mice of catalepsy-resistant strain. Comparison of Figs. 1 and 3 indicated a similarity between the effect of LPS on catalepsy and that on 5-HIAA/5-HT ratio in the midbrain and striatum. This result was not unexpected since the midbrain included the majority of the cell bodies of the ascending 5-HT neurons in the n. raphe dorsalis and n. raphe medianus (Jacobs and Azmitia, 1992), while 5-HT of the striatum was shown to be involved in catalepsy (Kulikov et al., 1992, 1995; Wadenberg, 1996). Recently a similar relationship between the effect of emotional stress on catalepsy and on 5HIAA/5-HT ratio in the midbrain was shown (Tikhonova et al., 2013). The brain 5-HT system in catalepsy-prone mice appeared to be more susceptible to LPS-induced and emotional stresses compared with catalepsy-resistant AKR mice. This results support a concept of the interaction between multiple alterations in the brain 5-HT system in the regulation of catalepsy. In conclusion, using original catalepsy-prone mice we first showed the involvement of genotype in the effect of LPS on catalepsy and the brain 5-HT system. We showed an association between the effect of LPS on hereditary catalepsy and that on 5-HT metabolism in the midbrain and striatum. The magnitudes of LPS-induced catalepsy and activation of 5-HT turnover in these brain structures were higher in catalepsy-prone than in catalepsy-resistant mice. This result indicated a possible involvement of the brain 5-HT system in the cataleptogenic effect of LPS. Catalepsy accompanied such grave mental disorders as schizophrenia, parkinsonism and extrapyramidal dysfunctions (Caroff et al., 2000; Daniels, 2009; Lee, 2007, 2010; Paparrigopoulos et al., 2009; Sanberg et al., 1988; Weder et al., 2008). Pinch-induced catalepsy was observed in the rats bred for high predisposition to audiogenic epilepsy (Surina et al., 2010). Hereditary catalepsy in mice met the face and predictive validity of a preclinical model of depression (Kulikov and
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Popova, 2008; Tikhonova et al., 2010). The association between the LPS induced catalepsy and 5-HT metabolism alterations can be interpreted as a construct validity of hereditary catalepsy as a prospective preclinical model of depression. Thus, hereditary catalepsy in CBA and D13 mice can be used as a model of the interaction between 5-HT and immune systems in the mechanisms of psychopathology. Acknowledgments This work was supported in part by the Russian Foundation for Basic Research (grant no. 11-04-00266-a), Molecular and Cell Biology Program of the Russian Academy of Sciences (project no. 6.7) and the grant no. 80.60 of the Ministry of Education and Science of the Russian Federation. References Amir S, Brown ZW, Amir Z, Ornstein K. Body-pinches induced long lasting cataleptic-like immobility in mice: behavioral characterization and the effects of naloxone. Life Sci 1981;10:1189–94. Bazovkina DV, Tibeikina MA, Kulikov AV, Popova NK. Effects of lipopolysaccharide and interleukin-6 on cataleptic immobility and locomotor activity in mice. Neurosci Lett 2011;487:302–4. Caroff SN, Mann SC, Keck Jr PE, Francis A. Residual catatonic state following neuroleptic malignant syndrome. J Clin Psychopharmacol 2000;20:257–9. Cho L, Tsunoda M, Sharma RP. Effects of endotoxin and tumor necrosis factor alpha on regional brain neurotransmitters in mice. Nat Toxins 1999;7:187–95. Daniels J. Catatonia: clinical aspects and neurobiological correlates. J Neuropsychiatry Clin Neurosci 2009;21:371–80. Dantzer R. Cytokine-induced sickness behavior: a neuroimmune response to activation of innate immunity. Eur J Pharmacol 2004;500:399–411. Dantzer R, O'Connor JC, Freund GG, Johnson RW, Kelley KW. From inflammation to sickness and depression: when the immune system subjugates the brain. Nat Rev Neurosci 2008;9:46–56. Dixon AK. Ethological strategies for defense in animals and humans: their role in some psychiatric disorders. Br J Med Psychol 1998;71:417–45. Dunn AJ. Endotoxin-induced activation of cerebral catecholamine and serotonin metabolism: comparison with interleukin-1. J Pharmacol Exp Ther 1992;261:964–9. Dunn AJ. Effects of cytokines and infections on brain neurochemistry. Clin Neurosci Res 2006;6:52–68. Dunn AJ, Chuluyan HE. Endotoxin elicits normal tryptophan and indolamine responses but impaired catecholamine and pituitary-adrenal responses in endotoxin-resistant mice. Life Sci 1994;54:847–53. Dunn AJ, Wang J. Cytokine effects on CNS biogenic amines. Neuroimmunomodulation 1995;2:319–28. Dunn AJ, Wang J, Ando T. Effects of cytokines on cerebral neurotransmission. Comparison with the effects of stress. Adv Exp Med Biol 1999;461:117–27. Givalois L, Becq H, Siaud P, Ixart G, Assenmacher I, Barbanel G. Serotoninergic and suprachiasmatic nucleus involvement in the corticotropic response to systemic endotoxin challenge in rats. J Neuroendocrinol 1999;11:629–36. Hsieh PF, Chia LG, Ni DR, Cheng LJ, Ho YP, Tzeng SF, et al. Behavior, neurochemistry and histology after intranigral lopopolysaccharide injection. Neuroreport 2002;13: 277–80. Jacobs BL, Azmitia EC. Structure and function of the brain serotonin system. Physiol Rev 1992;72:165–229. Kabiersch A, del Rey A, Honegger CG, Besedovsky HO. Interleukin-1 induces changes in norepinephrine metabolism in the rat brain. Brain Behav Immun 1988;2:267–74. Kostowski W, Gumulka W, Czlonkowski A. Reduced cataleptogenic effects of some neuroleptics in rats with lesioned midbrain raphe and treated with p-chlorophenylalanine. Brain Res 1972;48:443–6.
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Kulikov AV, Popova NK. Genetic control of catalepsy in mice. In: Torres S, Martin M, editors. Genetic predisposition to disease. New York: Nova Publishers; 2008. p. 215–36. Kulikov A, Kozlachkova E, Popova N. The activity of tryptophan hydroxylase in brain of hereditary predisposed to catalepsy rats. Pharmacol Biochem Behav 1992;43: 999–1003. Kulikov AV, Kozlachkova EYu, Maslova GB, Popova NK. Inheritance of predisposition to catalepsy in mice. Behav Genet 1993;23:379–84. Kulikov A, Kolpakov V, Maslova G, Kozintsev I, Popova N. Effect of selective 5-HT1A agonists and 5-HT2 antagonists on inherited catalepsy in rats. Psychopharmacology 1994;114:172–4. Kulikov A, Kozlachkova E, Kudryavtseva N, Popova N. Correlation between tryptophan hydroxylase activity in the brain and predisposition to pinch-induced catalepsy in mice. Pharmacol Biochem Behav 1995;50:431–5. Kulikov AV, Bazovkina DV, Kondaurova EM, Popova NK. Genetic structure of hereditary catalepsy in mice. Genes Brain Behav 2008;7:506–12. Kulikov AV, Sinyakova NA, Naumenko VS, Bazovkina DV, Popova NK. Association of glycoprotein gp130 with hereditary catalepsy in mice. Genes Brain Behav 2010;9:997–1003. Kulikov AV, Tikhonova MA, Kulikova EA, Volcho KP, Khomenko TM, Salakhutdinov NF, et al. A new synthetic varacin analogue, 8-(trifluoromethyl)-1,2,3,4,5benzopentathiepin-6-amine hydrochloride (TC-2153), decreased hereditary catalepsy and increased the BDNF gene expression in the hippocampus in mice. Psychopharmacology 2012;221:469–78. Lacosta S, Merali Z, Anisman H. Behavioral and neurochemical consequences of lipopolysaccharide in mice: anxiogenic-like effects. Brain Res 1999;818:261–303. Lavicky J, Dunn AJ. Endotoxin administration stimulates cerebral catecholamine release in freely moving rats as assessed by microdialysis. J Neurosci Res 1995;40:407–13. Lee JW. Catatonic variants, hyperthermic extrapyramidal reactions, and subtypes of neuroleptic malignant syndrome. Ann Clin Psychiatry 2007;19:9–16. Lee JW. Neuroleptic-induced catatonia: clinical presentation, response to benzodiazepines, and relationship to neuroleptic malignant syndrome. J Clin Psychopharmacol 2010;30:3–10. Linthorst AC, Flachskamm C, Muller-Preuss P, Holsboer F, Reut JM. Effect of bacterial endotoxin and interleukin-1 beta on hippocampal serotonergic neurotransmission, behavioral activity, and free corticosterone levels: an in vivo microdialysis study. J Neurosci 1995;15:2920–34. Merali Z, Lacosta S, Anisman H. Effects of interleukine-1 beta and mild stress on alterations of norepinephrine, dopamine and serotonin neurotransmission: a regional microdialysis study. Brain Res 1997;761:225–35. Naumenko EV, Popova NK, Starygin AG. Pituitary–adrenal system of animals in groups and in isolation. Zh Obshch Biol 1971;32:731–9. Paparrigopoulos T, Tzavellas E, Ferentinos P, Mourikis I, Liappas J. Catatonia as a risk factor for the development of neuroleptic malignant syndrome: report of a case following treatment with clozapine. World J Biol Psychiatry 2009;10:70–3. Popova NK, Gilinsky MA, Amstislavskaya TG, Morosova EA, Seif I, De Maeyer E. Regional serotonin metabolism in the brain of transgenic mice lacking monoamine oxidase A. J Neurosci Res 2001;66:423–7. Sanberg PR, Bunsey MD, Giordano M, Norman AB. The catalepsy test: its ups and downs. Behav Neurosci 1988;102:748–59. Surina NM, Fedotova IB, Kulikov AV, Poletaeva II. Pinch-induced catalepsy in rats of various genetic groups with different predisposition to audiogenic epilepsy. Zh Vyssh Nerv Deiat Im I P Pavlova 2010;60:364–71. Tikhonova MA, Alperina EL, Tolstikova TG, Bazovkina DV, Di VY, Idova GV, et al. Effects of chronic fluoxetine treatment on catalepsy and the immune response in mice with a genetic predisposition to freezing reactions: the roles of types 1A and 2A serotonin receptors and the tph2 and SERT genes. Neurosci Behav Physiol 2010;40:521–7. Tikhonova MA, Kulikov AV, Bazovkina DV, Kulikova EA, Tsybko AS, Bazhenova EYu, et al. Hereditary catalepsy in mice is associated with the brain dysmorphology and altered stress response. Behav Brain Res 2013;243:53–60. Valzelli L. The “isolation syndrome” in mice. Psychopharmacologia 1973;31:305–20. Wadenberg ML. Serotonergic mechanisms in neuroleptic-induced catalepsy in the rat. Neurosci Biobehav Rev 1996;20:325–39. Wang J, Dunn AJ. The role of interleukin-6 in the activation of the hypothalamo–pituitary– adrenocortical axis and brain indoleamines by endotoxin and interleukin-1 beta. Brain Res 1999;815:337–48. Weder ND, Muralee S, Penland H, Tampi RR. Catatonia: a review. Ann Clin Psychiatry 2008;20:97–107.
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On the association between lipopolysaccharide induced catalepsy and serotonin metabolism in the brain of mice genetically different in the predisposition to catalepsy Ekaterina Yu. Bazhenova ⁎, Alexander V. Kulikov, Maria A. Tikhonova, Daria V. Bazovkina, Daria V. Fursenko, Nina K. Popova Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, Novosibirsk 630090, Russian Federation
a r t i c l e
i n f o
Article history: Received 21 June 2013 Received in revised form 6 August 2013 Accepted 10 August 2013 Available online 28 August 2013 Keywords: Lipopolysaccharide Serotonin metabolism Catalepsy Brain Mice
a b s t r a c t The study of the interaction between nervous and immune systems in the mechanism of psychopathology is an important problem of neuroscience. Catalepsy (freezing reaction) is a passive defensive strategy in response to threatening stimuli. An exaggerated form of catalepsy is a syndrome of some grave mental disorders. Both the brain serotonin (5-HT) and immune systems were shown to be involved in the mechanism of catalepsy. Here we compared the effects of two doses (50 or 200 μg/kg, ip) of innate immune system activator, bacterial lipopolysaccharide (LPS), on catalepsy, 5-HT and its main metabolite, 5-hydroxyindole acetic acid (5-HIAA) in the hippocampus, striatum, and midbrain of mice of catalepsy-prone (CBA/Lac and AKR.CBA-D13Mit76) and catalepsyresistant (AKR/J) strains. The expression of LPS-induced catalepsy as well as 5-HIAA/5-HT ratio in the midbrain and striatum were significantly higher in mice of the catalepsy-prone strains compared with animals of the catalepsy-resistant strains. These results indicated an involvement of the brain 5-HT system in the cataleptogenic effect of LPS and open up new vistas for understanding the nervous–immune mechanism of behavioral disorders. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Catalepsy (tonic immobility, immobility reflex, animal hypnosis) is a state of pronounced motor inhibition characterized by a failure to correct an externally imposed, awkward posture. In some wild animals it is an element of natural freezing response to predator appearance (Dixon, 1998). An exaggerated form of catalepsy is the syndrome of grave mental disorders such as schizophrenia, parkinsonism and extrapyramidal dysfunctions (Caroff et al., 2000; Daniels, 2009; Lee, 2007, 2010; Paparrigopoulos et al., 2009; Sanberg et al., 1988; Weder et al., 2008). Drug-free catalepsy can be induced by pinching mice at the scruff of the neck (Amir et al., 1981). This pinch-induced catalepsy is a very rare phenomenon that is not found in mice of the most common inbred strains, such as C57BL/6J, DBA/2, AKR/J, etc. But about 50% of CBA/Lac mice showed catalepsy after several pinches (Kulikov et al., 1993). The main locus for catalepsy was mapped to the 61–70-cM fragment of chromosome 13 (Kulikov et al., 2008). Then, the CBA-derived 59–70 cM fragment of chromosome 13 containing the main locus
⁎ Corresponding author at: Laboratory of Behavioral Neurogenomics, Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, Lavrentyeva Ave. 10/2, 630090 Novosibirsk, Russian Federation. Tel.: +7 383 3636187; fax: +7 383 3331278. E-mail address: [email protected] (E.Y. Bazhenova). 0091-3057/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pbb.2013.08.009
of catalepsy transferred to the AKR genome and the recombinant AKR.CBA-D13Mit76 (D13) strain was created. A pronounced catalepsy was found in about 50% of D13 mice (Kulikov et al., 2008). Earlier we showed the involvement of the brain serotonin (5-HT) and innate immune systems in the regulation of pinch-induced catalepsy in mice. The key enzyme of 5-HT synthesis, tryptophan hydroxylase, in the striatum of mice of catalepsy-prone CBA strain was increased compared with mice of catalepsy-resistant AKR and C57BL/6 strains (Kulikov et al., 1995). 5-HT1A receptor agonist 8-OH-DPAT attenuated hereditary catalepsy in mice (Kulikov et al., 2012). Bacterial lipopolysaccharide (LPS) or proinflammatory interleukin-6 (IL-6) induced catalepsy in mice of catalepsy-resistant C57BL/6 strain (Bazovkina et al., 2011). Moreover, the gene encoding gp130 protein, a subunit of IL-6 receptor, is linked to hereditary catalepsy (Kulikov et al., 2008). Although, LPS increases 5-HT metabolism in the brain of rats (Dunn, 2006), the effect of LPS on 5-HT metabolism in mice is contradictory. On one hand, LPS increased the level of the main 5-HT metabolite, 5-hydroxyindoleacetic acid (5-HIAA) in the brain of C3H/He mice (Dunn and Chuluyan, 1994). On the other hand LPS produced no effect on 5-HT metabolism in the brain of BALB/c mice (Cho et al., 1999). A possible genotype influence on the effect of LPS on the brain 5-HT system could be hypothesized. In order to test this hypothesis we studied the effect of LPS on the brain 5-HT metabolism and catalepsy in mice of catalepsy-resistant and catalepsy-prone strains.
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2. Methods 2.1. Animals and treatments The experiments were carried out on adult male mice (3– 4 months old, weighting 28 ± 4 g) of catalepsy-resistant AKR/J (n = 51), as well as catalepsy-prone CBA/Lac (n = 50) and recombinant AKR.CBAD13Mit76 (D13) (n = 51) strains. The D13 strain was created in the Institute of Cytology and Genetics by transferring the 59–70 cM fragment marked with microsatellites D13Mit74 (59 cM), D13Mit76 (61 cM) and D13Mit214 (71 cM) of chromosome 13 containing the major gene of catalepsy from CBA/Lac to the genome of AKR/J strain. D13 and AKR have the same genetic background and are distinguished by the CBA- or the AKR-derived 59–70 cM fragment, respectively. However, about 50% of D13 mice show CBA-like catalepsy, while none of AKR mice displays catalepsy (Kulikov et al., 2008). AKR, CBA and D13 strains were maintained at the Institute of Cytology and Genetics by brother–sister inbreeding for at least 55, 55 and 20 generations, respectively. After weaning, mice were separated by sex and kept six animals per cage (40 × 30 × 15 cm) until the age of 3–4 months 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 individually housed in cages of the same size to eliminate group effect. This two-day period of separation was shown to be sufficient to eliminate the group effect (Naumenko et al., 1971), but short to produce an isolation effect (Valzelli, 1973). 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. LPS (Escherichia coli 055:B5; Sigma-Aldrich Inc., St. Louis, MO, USA) was diluted in sterile saline and injected intraperitoneally in the doses of 50 or 200 μg/kg 3 h prior to behavioral test or decapitation. It was shown that the peak of sickness behavior was observed in 2–6 h after an ip LPS administration (Dantzer et al., 2008). These doses were chosen since earlier we showed that 200 μg/kg of LPS decreased locomotion and social behavior in D13 and AKR mice, while 50 μg/kg of LPS was effective only in D13 mice (Kulikov et al., 2010).
of 7 animals. Three hours after LPS or saline treatment the animals were decapitated and their striatum (caudate nucleus, putamen and globus pallidus), hippocampus (dentate gyrus, CA1 and CA3) and midbrain (n. raphe dorsalis and n. raphes medianus) were dissected, frozen with liquid nitrogen and kept at −70 °C until the measurement. Concentrations of 5-HT and 5-HIAA were assessed with high performance liquid chromatography (HPLC) (Kulikov et al., 2012). Tissue samples were homogenized in 200 μl of buffer containing 0.4 M HClO4 (Sigma, USA) and 0.27 mM EDTA (Ameresco, USA). The homogenates were centrifuged 5 min at 15000 g (4 °C) and supernatant was filtered through Whatman GF/C fiberglass filters (Whatman Int. Ltd., UK). Concentrations of 5-HT and 5-HIAA were then evaluated by HPLC on Nucleosil C8 column (Nucleosil C8 column, 3 μm particle size, L × I.D. 100 mm × 4.6 mm, Sigma-Aldrich, USA) with electrochemical detection (500 mV, Coulochem III, ESA, Inc., USA) and flowcell (BASInc, USA) using solvent delivery module LC-20AD (Shimadzu Corporation, Japan). The mobile phase contained NaH2PO4 (100 mM, pH = 4.5), 0.1 mM Na2EDTA, 1.4 mM 1-octanesulfonic acid sodium salt (Sigma, USA) and methanol (4 vol.%; Vekton Ltd., Russia). Its flow rate was 0.6 ml/min. Standard solution containing 2 ng of 5-HT and 5-HIAA was repeatedly assayed throughout the entire procedure. The concentration of 5-HT and 5-HIAA peaks was estimated by comparison of the magnitudes of corresponding picks with the respective external standards using the MultiChrom v.1.5 software (Ampersand Ltd., Russia) and expressed in μg per g of tissue sample. The intensity of 5-HT metabolism was evaluated by the 5-HIAA/5-HT ratio. The 5-HIAA/5-HT ratio reflects adequately the intensity of oxidative deamination of 5-HT and is more valid than the commonly used 5-HIAA level (Popova et al., 2001).
2.4. Statistics All values were presented as means ± SEM and compared with two-way analysis of variance (ANOVA) followed by the Fisher's post hoc analysis.
2.2. Test for catalepsy
3. Results
The effect of LPS (50 or 200 μg/kg) on catalepsy was tested in AKR (n = 10 for 50 μg/kg and n = 11 for 200 μg/kg), CBA (n = 8 for 50 μg/kg and n = 10 for 200 μg/kg) and D13 (n = 11 for 50 μg/kg and n = 9 for 200 μg/kg) mice. The control animals (9 AKR, 11 CBA and 10 D13) were injected with saline. Catalepsy was tested according to an earlier described procedure (Kulikov et al., 1993, 2008). Animals were pinched with two fingers for 5 s 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 s of freezing. Immobility time of more than 20 s was considered as positive (cataleptic) response. Every animal was successively tested with 2-min intervals (the mouse was placed in its home cage between the trials) until three positive responses were achieved, but no more than 10 times. Immobility time was calculated as the mean of three trials with the maximal values. The recovery of this procedure was about 80% (Kulikov et al., 1993).
3.1. Effect of LPS on catalepsy
2.3. Measurement of 5-HT metabolism The effects of LPS (50 or 200 μg/kg) on the concentration of 5-HT and its main catabolite — 5-hydroxyindoleacetic acid (5-HIAA) in the brain were studied in nine groups (three strains × three treatments)
Catalepsy was shown in 1 out of 10 saline-treated AKR mice, in 9 out of 10 control D13 animals and in 8 out of 11 control CBA mice. 50 μg/kg of LPS did not produce catalepsy in AKR mice (only 1 of 10 LPS-treated AKR showed catalepsy), but catalepsy was observed in all 11 D13 mice and 8 CBA mice treated with LPS. Eight out of 11 AKR as well as all D13 and CBA mice treated with 200 μg/kg showed catalepsy. Significant effects of genotype (F2,80 = 53.15, p b 0.001), LPS (F2,80 = 14.9, p b 0.001) and genotype × LPS interaction (F4,80 = 4.05, p b 0.01) on catalepsy duration were shown. Immobility time in the saline-treated D13 or CBA mice was higher than in AKR mice (p b 0.01). LPS at the dose of 50 μg/kg did not affect the time of catalepsy in AKR mice, but significantly increased it in D13 mice (from 72.03 ± 11.43 s in saline-treated to 115.9 ± 2.3 s in LPS-treated, p b 0.001; Fig. 1) and CBA mice (from 46.18 ± 8.7 s in saline-treated to 96.25 ± 9.91 s in LPS-treated, p b 0.001; Fig. 1). LPS at the dose of 200 μg/kg increased the time of catalepsy in all strains: AKR (p b 0.01), D13 (p b 0.05) and CBA (p b 0.001) (Fig. 1). No difference in catalepsy duration between CBA and D13 mice treated saline, 50 μg/kg or 200 μg/kg of LPS was revealed (p N 0.05). At the same time, immobility time in catalepsy-resistant AKR mice treated with saline (p b 0.01 vs CBA and p b 0.001 vs D13), 50 μg/kg (p b 0.0001) or 200 μg/kg (p b 0.01 vs CBA and p b 0.001 vs D13) of LPS was significantly lower compared with catalepsy-prone CBA or D13 mice.
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3.2. Effect of LPS on 5-HT metabolism
Fig. 1. Mean catalepsy duration (s) in the saline- and LPS-treated mice of catalepsy-resistant (AKR) and catalepsy-prone (CBA and D13) strains. All animals were ip injected with saline or LPS 3 h before the test. The numbers of mice: 1) AKR: saline — 9, 50 μg/kg of LPS — 10, 200 μg/kg of LPS — 11; 2) CBA: saline — 11, 50 μg/kg of LPS — 8, 200 μg/kg of LPS — 10; 3) D13: saline — 10, 50 μg/kg of LPS — 11, 200 μg/kg of LPS — 9. *p b 0.05, **p b 0.01, ***p b 0.001 vs corresponding saline-treated control; ##p b 0.01, ###p b 0.001 vs salinetreated AKR; &&&p b 0.001 vs LPS-treated (50 μg/kg) AKR; @@@p b 0.001 vs LPS-treated (200 μg/kg) AKR.
No effect of genotype on 5-HT level in the hippocampus (F2,54 b 1) and the midbrain (F2,54 b 1) was shown. At the same time, a significant effect of genotype on 5-HT level in the striatum was revealed (F2,54 = 3.5, p b 0.05): 5-HT concentration in this structure was higher in AKR mice compared with CBA (p = 0.052) and D13 (p b 0.01) mice. LPS decreased 5-HT level in the hippocampus (F2,54 = 7.6, p b 0.01) and striatum (F2,54 = 22.7, p b 0.001), but increased 5-HT concentration in the midbrain (F2,54 = 4.4, p b 0.05). LPS at the dose of 50 μg/kg significantly decreased 5-HT level in the hippocampus of AKR (p b 0.01) and D13 (p b 0.05) mice. At the same time, 200 μg/kg of LPS significantly decreased 5-HT level in the striatum of mice of the studied strains. LPS at the dose of 200 μg/kg (p b 0.05), but not 50 mg/kg increased 5-HT level in the midbrain of mice of CBA strain compared with the control mice. A tendency to increase was found in congenic D13 mice at both doses of LPS (p = 0.055 and p = 0.054) (Fig. 2A). Neither genotype (F2,54 = 2.0, p N 0.05), nor LPS (F2,54 = 3.1, p N 0.05) altered 5-HIAA level in the hippocampus. No difference in 5-HIAA concentration in the striatum (F2,54 b 1) or midbrain (F2,54 = 1.2, p N 0.05) of the saline-treated animals was found. At the same time, LPS significantly altered 5-HIAA level in these structures (F2,54 =
Fig. 2. 5-HT (A) and 5-HIAA (B) levels (μg/g of wet tissue mass) in the midbrain, hippocampus and striatum of the saline- and LPS-treated mice of catalepsy-resistant (AKR) and catalepsyprone (CBA and D13) strains. All animals were ip injected with saline or LPS 3 h before decapitation. The bars represent means ± SEM of seven values. *p b 0.05, **p b 0.01, ***p b 0.001 vs corresponding saline-treated control; #p b 0.05, ##p b 0.01 vs saline-treated AKR.
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3.6, p b 0.05 in the striatum and F2,54 = 11.3, p b 0.001 in the midbrain). While LPS at the dose of 200 μg/kg significantly decreased 5-HIAA in the striatum of AKR mice (p = 0.02), both doses of LPS significantly increased 5-HIAA level in the midbrain of CBA (p b 0.01 at 50 μg/kg and p b 0.001 at 200 μg/kg) and D13 (p b 0.01) mice (Fig. 2B). LPS increased 5-HT turnover (5-HIAA/5-HT rate) in the midbrain (F2,54 = 13.2, p b 0.001), hippocampus (F2,54 = 45.4, p b 0.001) and striatum (F2,54 = 12.2, p b 0.001). Both doses of LPS significantly increased 5-HT turnover in the hippocampus of mice of the three strains studied (p b 0.001). LPS at the dose of 50 μg/kg increased 5-HT turnover in the midbrain only of the catalepsy-prone CBA (p b 0.001) and D13 (p b 0.05) strains without any effect on this index in AKR mice. At the same time, 200 μg/kg of LPS significantly increased 5-HT turnover in this structure in all strains. Neither 50 μg/kg, nor 200 μg/kg of LPS affected 5-HT turnover in the striatum of AKR mice. However, LPS at the dose of 200 μg/kg significantly increased 5-HT metabolism in the striatum of catalepsy-prone CBA (p b 0.01) and D13 (p b 0.05) mice (Fig. 3). 4. Discussion Earlier we found that LPS (100 or 200 μg/kg) or IL-6 (7.5 or 10.0 μg/kg) induced catalepsy in mice of catalepsy-resistant C57BL/ 6 strain (Bazovkina et al., 2011). Here, we confirmed our earlier data and showed that 200 μg/kg of LPS produced catalepsy in mice of another catalepsy-resistant AKR strain. An important result of the present study was that LPS produced more pronounced cataleptogenic effect on mice of catalepsy-prone CBA and D13 strains than on mice of catalepsyresistant AKR strain. Although mice of AKR and D13 strains had the same AKR-derived genetic background (Kulikov et al., 2008), they differed in the cataleptogenic effect of the low dose of LPS (50 μg/kg). This dose failed to induce catalepsy in AKR mice, but significantly increased the time of cataleptic immobility in D13 and CBA mice. Since D13 and CBA mice have the same CBA-derived 59–70 cM fragment of chromosome 13 containing the main locus of catalepsy (Kulikov et al., 2008), this result indicated an association between the main locus of catalepsy and the cataleptogenic effect of LPS. An intriguing question concerns the mechanism of this association between hereditary catalepsy and the cataleptogenic effect of LPS. There are data that show that the brain 5-HT system is involved in the mechanism of catalepsy in rats. Agonists of 5-HT1A receptor dramatically reduced neuroleptic-induced (Wadenberg, 1996) and hereditary (Kulikov et al., 1994) catalepsy. Blockade of 5-HT synthesis with pCPA, an irreversible inhibitor of the key enzyme of 5-HT synthesis, tryptophan hydroxylase 2 (TPH2), significantly reduced both neuroleptic-
Fig. 3. 5-HIAA/5-HT ratio in the midbrain, hippocampus and striatum of the saline- and LPS-treated mice of catalepsy-resistant (AKR) and catalepsy-prone (CBA and D13) strains. All animals were ip injected with saline or LPS 3 h before decapitation. The bars represent means ± SEM of seven values. *p b 0.05, **p b 0.01, ***p b 0.001 vs corresponding salinetreated control.
induced (Kostowski et al., 1972) and hereditary catalepsy (Kulikov et al., 1992). Selective breeding for catalepsy increased TPH2 activity in the striatum of rats of Genetic Catalepsy (GC) strain (Kulikov et al., 1992). At the same time, the association between hereditary catalepsy and 5-HT system in mice is less clear. The only study showed a significant increase of TPH2 activity in the striatum of catalepsy-prone CBA mice compared to such catalepsy-resistant strains as AKR/J and C57BLl/6J (Kulikov et al., 1995). LPS increases blood levels of such proinflammatory cytokines as ILβ, IL-6, and TNFα (Dantzer, 2004). The data on the effects of LPS and IL-1β on 5-HT metabolism in the brain are rather contradictory. Majority of authors showed that LPS or IL-1β increased 5-HIAA concentration in rat brain (Dunn, 1992; Dunn and Wang, 1995; Dunn et al., 1999; Givalois et al., 1999; Hsieh et al., 2002; Lacosta et al., 1999; Lavicky and Dunn, 1995; Linthorst et al., 1995; Merali et al., 1997; Wang and Dunn, 1999). However, some authors did not find the effect of IL-1β on 5-HT metabolism (Kabiersch et al., 1988). In mice, the effect of LPS on 5-HT metabolism seems to depend on genotype: LPS increased 5HIAA level in the brain of C3H/HeJ and C3H/HeN mice (Dunn and Chuluyan, 1994), but not in BALB/c mice (Cho et al., 1999). At the present study, we found that in all studied strains LPS decreased 5-HT level in the hippocampus and striatum where the 5-HT endings are located and increased the level of 5-HT in the midbrain where the 5-HT bodies are located. At the same time, LPS significantly increased 5-HIAA level in the midbrain of mice of catalepsy-prone CBA and D13 strains only. LPS increased 5-HIAA/5-HT ratio in the hippocampus of all strains studied in a genotype-independent manner. At the same time, in the midbrain and striatum the effect of LPS on this trait depended on genotype: it was more prominent in mice of catalepsyprone strains than in animals of catalepsy-resistant strain. In a steadystate condition when all synthesized 5-HT was metabolized to 5-HIAA, an increase in 5-HIAA/5-HT ratio corresponds to activation of 5-HT metabolism. Therefore, the present results indicated that LPS produced more intensive effect on 5-HT metabolism in the midbrain and striatum of mice of genetically predisposed to catalepsy strains compared with mice of catalepsy-resistant strain. Comparison of Figs. 1 and 3 indicated a similarity between the effect of LPS on catalepsy and that on 5-HIAA/5-HT ratio in the midbrain and striatum. This result was not unexpected since the midbrain included the majority of the cell bodies of the ascending 5-HT neurons in the n. raphe dorsalis and n. raphe medianus (Jacobs and Azmitia, 1992), while 5-HT of the striatum was shown to be involved in catalepsy (Kulikov et al., 1992, 1995; Wadenberg, 1996). Recently a similar relationship between the effect of emotional stress on catalepsy and on 5HIAA/5-HT ratio in the midbrain was shown (Tikhonova et al., 2013). The brain 5-HT system in catalepsy-prone mice appeared to be more susceptible to LPS-induced and emotional stresses compared with catalepsy-resistant AKR mice. This results support a concept of the interaction between multiple alterations in the brain 5-HT system in the regulation of catalepsy. In conclusion, using original catalepsy-prone mice we first showed the involvement of genotype in the effect of LPS on catalepsy and the brain 5-HT system. We showed an association between the effect of LPS on hereditary catalepsy and that on 5-HT metabolism in the midbrain and striatum. The magnitudes of LPS-induced catalepsy and activation of 5-HT turnover in these brain structures were higher in catalepsy-prone than in catalepsy-resistant mice. This result indicated a possible involvement of the brain 5-HT system in the cataleptogenic effect of LPS. Catalepsy accompanied such grave mental disorders as schizophrenia, parkinsonism and extrapyramidal dysfunctions (Caroff et al., 2000; Daniels, 2009; Lee, 2007, 2010; Paparrigopoulos et al., 2009; Sanberg et al., 1988; Weder et al., 2008). Pinch-induced catalepsy was observed in the rats bred for high predisposition to audiogenic epilepsy (Surina et al., 2010). Hereditary catalepsy in mice met the face and predictive validity of a preclinical model of depression (Kulikov and
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