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Catalepsy or pronounced freezing is a natural passive defense strategy in animals and a syndrome of some mental disorders in human. Hereditary catalepsy was shown to be associated with depressive-like features in rats and mice. The loci underlying the difference in predisposition to catalepsy between catalepsy-prone CBA/lacJ and catalepsy-resistant AKR/J mice were mapped using congenic line and selective breeding approaches. Three congenic mouse lines (AKR.CBA-D13Mit76C, AKR.CBA-D13Mit76A and AKR.CBA-D13Mit78) carrying the 59- to 70-, 61- to 70- and 71- to 75-cm fragments of chromosome 13 transferred from the CBA to the AKR genome were created by nine successive backcrossing of (CBA × AKR)F1 on AKR strain. Because catalepsy was found only in the AKR.CBA-D13Mit76C and AKR.CBA-D13Mit76A mice, the major gene of catalepsy was mapped on the fragment of 61–70 cm. Selective breeding of the (CBA × (CBA × AKR))BC backcross generation for high predisposition to catalepsy showed numerous genome-wide distributed CBA-derived alleles as well as the AKR-derived alleles mapped on chromosome 17 and on the proximal parts of chromosomes 10 and 19 that increased the cataleptogenic effect of the major gene.
A pronounced catalepsy in rats and mice induced by administration of haloperidol is considered an animal model of extrapyramidal syndrome (Klemm 1989; Sanberg et al. 1988). Hereditary catalepsy in rats of the catalepsy-prone genetic catalepsy (GC) strain is accompanied with multiple behavioral, endocrine and neurochemical alterations observed in depressive patients (Kulikov et al. 2006). Moreover, catalepsy in GC rats is sensitive only to chronic but not acute treatment with classic antidepressant imipramine resembling the dynamic of therapeutic drug effect (Kulikov et al. 2004).
Prolonged immobility induced by pinching mice at the scruff of the neck is another model of hereditary catalepsy (pinch-induced catalepsy) (Amir et al. 1981). A significant interstrain difference in the predisposition to pinch-induced catalepsy was shown: freezing reaction was found in 54% of males and females of CBA mouse strain but it was never detected in animals of AKR strain (Kulikov et al. 1993). Mice of antidepressant-sensitive catalepsy (ASC) line selectively breeding for high predisposition to catalepsy from the (CBA × (CBA × AKR))BC backcross population between catalepsy-prone CBA and catalepsy-resistant AKR strains showed depressive-like behavioral alterations similar to those found in GC rats (Bazovkina et al. 2005). Moreover, catalepsy in ASC mice like that in GC rats was sensitive to chronic but not acute treatment of imipramine (Tikhonova et al. 2006).
Therefore, the genetic mechanism underlying hereditary catalepsy is undoubtedly an important problem of behavioral genetics.
Single marker mapping using (CBA × (CBA × AKR))BC backcrosses and 65 genome-wide distributed polymorphic microsatellites found one major locus on the distal fragment of chromosome 13 (Kulikov et al. 2003). Interval mapping with six microsatellites positioned the major gene of catalepsy on the 47- to 75-cm fragment of chromosome 13 (Kulikov & Bazovkina 2003). However, these two commonly-used Quantitative Trait Loci analysis methods were developed for mapping quantitative traits and are difficult to apply to binary traits (Brodkin et al. 2002).
The main aim of the present paper was to map the genes encoding predisposition to catalepsy in mice using congenic lines and selective breeding experiment. It was intended to compare catalepsy in the congenic lines created by transferring the fragments of chromosome 13 from the CBA to the AKR genome and to study the effect of selective breeding for catalepsy on the distribution of CBA- and AKR-derived fragments in the mouse genome.
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The number of cataleptic males and females in the AKR.CBA-D13Mit76 and AKR.CBA-D13Mit78 lines are shown in Table 1. Because no sexual difference in the percentage of cataleptics was found (χ2 < 1, P > 0.05), the data on males and females were pooled together for further analysis. There were no differences in the percentage of cataleptics in the AKR.CBA-D13Mit76C and AKR.CBA-D13Mit76A sublines (52.3 and 52%, χ2 < 1, P > 0.05). At the same time, none of the 70 tested AKR.CBA-D13Mit78 mice showed catalepsy (χ2= 52, P < 0.001). The data unambiguously indicated that the main gene of catalepsy was mapped on the 61- to 70-cm fragment of chromosome 13 (Fig. 2).
Table 1. Number of catalepsy-prone and -resistant AKR.CBA-D13Mit76 and AKR.CBA-D13Mit78 mice
|Genotype||Sex||Catalepsy-prone mice*||Catalepsy-resistant mice|
Figure 2. The percentage of cataleptics in CBA, AKR, AKR.CBA-D13Mit76C, AKR.CBA-D13Mit76A and AKR.CBA-D13Mit78 mice. The AKR and CBA fragments of chromosome 13 are dark and white, respectively.
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Thirty-three (71.7%) cataleptics were found among 46 males of ASC mice chosen to study the effect of selective breeding. The distribution of the CBA and AKR alleles in the ASC males was shown in Fig. 3 and Table 2. The breeding did not significantly affect the initial (3:1) distribution for 14 markers located on chromosomes 2 and 5, proximal parts of chromosomes 3, 7, 8, 12 and 13 as well as the distal parts of chromosomes 9, 14 and 15. At the same time, the breeding increased the concentrations of CBA-derived alleles for 24 microsatellites on chromosomes 1, 4, 6 and 14, the proximal parts of chromosomes 9, 11 and 15 as well as the distal parts of chromosomes 3, 7, 8, 13, 18 and 19. A significant increase of the level of AKR-derived alleles for seven markers on chromosomes 10, 12, 16, 17, 18 and 19 was found.
Figure 3. Distribution of the CBA- and AKR-derived alleles of 45 microsatellites in 46 males representing 46 different ASC families. The horizontal lines mark the microsatellite positions on the chromosomes. The fragment size was assumed to be 10 cm and includes corresponding microsatellites. The fragments unaffected by breeding are not marked. The distributions of CBA and AKR alleles of these fragments in the ASC families can be explained by drift. The CBA- and AKR-derived fragments whose concentrations were significantly increased by selective breeding compared with those predicted by drift are marked dark and white, respectively.
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Table 2. Effect of selective breeding for catalepsy on the distribution of CBA- and AKR-derived alleles of the 45 polymorphic markers in 46 males of ASC mice
|Chromosome||Marker||Distance, cm||CBA-derived allele||AKR-derived allele||χ2|
Comparison of the distributions of CBA- and AKR-derived alleles of 45 microsatellites in 46 ASC families with those obtained from the simulation experiment indicated that the observed increase of 24 CBA- and seven AKR-derived alleles did not result from genetic drift. The numbers of these 24 CBA- and seven AKR-derived alleles (Table 2) were higher than those of the respective maximal values predicted from genetic drift (82 for CBA and 36 for AKR).
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Earlier, a single-gene hypothesis of inheritance of the predisposition to pinch-induced catalepsy was proposed because (CBA × AKR)F1 hybrids never displayed freezing, and the number of cataleptics in (CBA × AKR)F2 intercross was in a good agreement with the expected 3:1 ratio (Kulikov et al. 1993). This supposition was confirmed recently by the demonstration that the number of cataleptics in (CBA × (CBA × AKR))BC backcross population did not differ from the expected 1:1 ratio (Kondaurova et al. 2006). The single marker analysis carried out on the backcrosses between catalepsy-prone CBA and catalepsy-resistant AKR strains mapped the main gene of catalepsy on the distal fragment of chromosome 13 (Kulikov et al. 2003). Further interval mapping located the major gene position on the 47- to 75-cm fragment of the chromosome. The main factor defined about 20% of catalepsy penetrance, while other 31% of the trait variability was controlled by polygenes (Kulikov & Bazovkina 2003). This interval included the gene encoding 5-HT1A serotonin receptor (58 cm). Recently, it was shown that selective breeding for catalepsy eliminated the AKR-derived allele of D13Mit76 linked to the 5-HT1A serotonin receptor gene (Kondaurova et al. 2006). Because agonists of the receptor attenuated neuroleptic-induced (Broekkamp et al. 1988; Invernizzi et al. 1988; Neal-Beliveau et al. 1993; Wadenberg 1996; Wadenberg et al. 1999) and hereditary (Kulikov et al. 1994; Popova et al. 1994) catalepsy, the 5-HT1A receptor gene was proposed as the most probable candidate gene for predisposition to catalepsy in mice (Kondaurova et al. 2006).
In the current study, the major gene of catalepsy was mapped using AKR.CBA-D13Mit76A, AKR.CBA-D13Mit76C and AKR.CBA-D13Mit78 congenic lines carrying, respectively, the 61- to 70-, 59- to 70- and 71- to 75-cm CBA-derived fragments of chromosome 13 transferred on the AKR genome. A pronounced catalepsy was found in half of the AKR.CBA-D13Mit76A and AKR.CBA-D13Mit76C mice. At the same time, no AKR.CBA-D13Mit78 mice showed immobility. The data indicate that the major gene of catalepsy is located on the 61- to 70-cm fragment of chromosome 13.
The recent results confirmed the linkage of catalepsy to D13Mit76 microsatellite but rejected the hypothesis about association between hereditary catalepsy and 5-HT1A receptor gene. Although the AKR.CBA-D13Mit76A mice received the 5-HT1A receptor gene from AKR strain, their predisposition to catalepsy was similar to that of AKR.CBA-D13Mit76C carrying the CBA-derived receptor gene.
The 61- to 70-cm (111- to 116-Mb) fragment of chromosome 13 contains 34 protein-coding genes (Ensemble, http://www.ensembl.org/Mus_musculus). Information about the expression in mouse brain for nine of these genes is not available. Among other 25 genes, only eight are highly expressed in mouse brain (GNF SymAtlas, http://symatlas.gnf.org/SymAtlas). They are Gpbp1 (vasculin), Map3k1 [microtubule-associated protein (MAP) kinase kinase kinase 1], Rpl41 (ribosomal protein L41), Il6st (gp130 signal transducer), Ppap2a (phosphatidic acid phosphatase 2a), Gzmk (granzyme K), Snag1 (sorting nexin-associated golgi protein 1) and Hspb3 (heat shock protein 3). Because no information about the neurobiological significance of the proteins encoded by the Rpl41, Ppap2a and Snag1 was available, these genes were excluded from the list of putative candidate genes. At the same time, the proteins encoded by the other genes seemed to be involved in the regulation of important cellular functions. The new protein, vasculin, encoded by the Gpbp1 gene, might play an important role in vascular biology (Bijnens et al. 2003). It can modify behavior throughout the regulation of brain blood vessels. The kinase of MAP kinase kinase is an enzyme of the cellular signal transduction from numerous hormones, growth factors and cytokines, and it participates in the regulation of numerous cellular functions (Robinson & Coob 1997). The Il6st gene codes the gp130 protein, which is the shared protein in the signal transduction from ciliary neurotrophic factor, leukemia inhibitor factor, oncostatin M and interleukins 6 and 11, participating in the cell differentiation, immune and endocrine regulation (Chesnokova & Melmed 2002). The granzyme K was shown to be involved in the mechanism of apoptosis (Zhao et al. 2007). Although the function of heat shock protein 3 is still not known, it seems to participate in the mechanisms of cell survival like other proteins of the shock proteins family (Suglyama et al. 2000). So, alterations in each of these five genes could result in such behavioral dysfunction as catalepsy. It is worthy to note that these five putative candidate genes regulate general intracellular functions, such as signal transduction and apoptosis. At the same time, no gene regulating the function of specific neurotransmitters involved in the mechanisms of freezing was detected in the 61- to 70-cm fragment of chromosome 13 linked to catalepsy.
The general genetic mechanisms predict that the combination of long-term selection with brother–sister inbreeding will increase the concentrations of the genes facilitating freezing, while the concentrations of the loci that do not link to catalepsy will not be altered by the selection process from their initial concentration (in the backcross population), namely 75% of CBA-derived and 25% of AKR-derived populations. It was calculated (Falconer 1960, p. 91) that 15 generations of such inbreeding were sufficient to ensure the probability of 0.925 of allele fixation in the ASC line. So, the distribution of CBA- and AKR-derived fragments in the ASC families can detect numerous loci increasing the predisposition to catalepsy. Here, we showed that the long-term selection (for eight generations) significantly altered the concentrations of 31 polymorphic microsatellite alleles and did not change the rates of other 14 alleles in the ASC line compared with those in the initial backcross population. Then the selection-induced alterations of the CBA- and AKR-derived allele concentrations were fixed in the ASC families during 15 successive generations of brother–sister inbreeding. Therefore, the observed significant increase of the concentrations of 24 CBA-derived fragments on chromosomes 1, 3, 4, 5, 6, 7, 8, 9, 11, 13, 14, 15, 18 and 19 as well as seven AKR-derived fragments on chromosomes 10, 12, 16, 17, 18 and 19 compared with those expected (75:25) indicated that they contained genes facilitating freezing and were conserved during the selective breeding for catalepsy. The most impressive result was the increase of the concentration of the AKR-derived fragments in the ASC mice, suggesting that catalepsy-resistant AKR mice carried alleles increasing the cataleptogenic effect of the CBA-derived allele of the major gene.
Using interval mapping, we showed that the main gene of catalepsy controlled 20% of the trait penetrance, while other 31% of the trait variability resulted from numerous polygenes (Kulikov & Bazovkina 2003). The congenic AKR.CBA-D13Mit76 and selected ASC lines markedly improved this evaluation. The cataleptogenic effects of the major gene on chromosome 13 as well as the polygenes were approximated with comparison of the percentage of cataleptics in CBA, ASC and AKR.CBA-D13Mit76 mice. The observed increase (21%) of the percentage of cataleptics in ASC (72%) compared with CBA mice (51%) results from the AKR-derived alleles of the polygenes mapped on chromosomes 10, 17 and 19. Because the percentage of cataleptics in AKR.CBA-D13Mit76 mice (52%) is the sum of effects caused with the major gene and the AKR-derived alleles, the predicted effect of the former is about 31% (=52 − 21%). The difference between the percentage of cataleptics in CBA mice and the major gene effect (51 − 31% = 20%) gives an estimation of the total effect of the CBA-derived alleles of polygenes (20%). These estimations of the effects of the main gene and polygenes are close to the values obtained with interval mapping (Kulikov & Bazovkina 2003).
Therefore, the congenic line models allow reducing the interval of the major gene position down to 10 cm compared with that obtained using QTL analysis (28 cm). Furthermore, the selective breeding approach found other 29 genome-wide distributed polygenes undetectable with QTL analysis.
The genetic structure of hereditary catalepsy in mice includes one major gene mapped on the fragment of 61–70 cm on chromosome 13 that determines approximately 31% of the trait penetrance and 29 genome-wide distributed genes – modifiers – producing the total effect on catalepsy to about 41%. Catalepsy was shown only in homozygous for the CBA allele of the major gene mice. Although the minor genes – modifiers – were unable to induce catalepsy along, they increased significantly the cataleptogenic effect of the CBA alleles of the major gene.
Haloperidol-induced catalepsy in mice was linked to the gene encoding dopamine D2 receptor (chromosome 9) (Kanes et al. 1996; Patel & Hitzemann 1999) that was the molecular target for haloperidol. On the contrary, catalepsy in mice is not linked to D2 receptor gene and it seems to be regulated with different genetic and molecular mechanisms. Although the involvement of D2 receptor in the regulation of the catalepsy is expected, the observed high predisposition to catalepsy in CBA and ASC mice does not result from mutation in the gene encoding this receptor.