The remote effects of neonatal injections of caffeine and piracetam on audiogenic seizure susceptibility in mice of three genotypes

Neonatal DBA/2J, 101/HY and CBA/Lac/Sto mice (2-7-day-old) were subcutaneously injected with caffeine (200 mg/kg), piracetam (50 mg/kg) or distilled water. At the age of 1 month, they were tested for audiogenic seizure susceptibility (SS). The neonatal injections changed SS in 1-month-old mice in a genotype-dependent manner. Distilled water (control of neonatal pain stimulation) slightly reduced the audiogenic fit severity (arbitrary scores) the effect being most distinct in DBA/2J, less strong in 101/HY strain and absent in CBA. Caffeine neonatal injections induced slight changes in DBA/2J, no changes in CBA and increased SS in 101/HY mice. Piracetam reduced fit intensity in DBA/2J mice but increased it in CBA and, especially, in 101/HY strain. Genotype-dependent differences in physiological mechanisms of audiogenic seizures may be responsible for different remote effects of early treatment.     

Neonatal Semax and saline injections induce open-field behavior changes in mice of different genotypes

DBA/2, CBA mice, and their F1 hybrids (first series) and 101/HY and C3H mice (second series) were injected as neonates (2-7 days of life) with Semax (sc., 7 microg per animal). Semax is a peptide analogue of ACHT4-10 fragment which is resistant to degradation. The common feature of remote effects of both Semax and saline injections was the set of changes in the open-field behavior in adult (2.5- to 3-month-old) animals as compared to intact mice. Unexpectedly, the neonatal saline injections induced many changes in adult behavior, part of these effects being genotype-dependent. The most conspicuous shifts (genotype-dependent increase or decline) in freezing, grooming and rearing scores were displayed by DBA/2 and C3H mice, whereas the hole-poke frequencies were significantly changed in CBA and C3H mice. Squares crossed in the center of arena and rearing number were significantly increased in saline group of DBA/2 mice, whereas in Semax-injected DBA/2 group they were approximately equal to the level of intact mice. This means that the remote effects of noxious stimulation (injections of saline) were in some ways "compensated" as the result of concomitant peptide effect. At the same time, the numbers of freezing and grooming episodes were also increased in these groups. Because exploratory behavior and manifestations of anxiety increased or decreased simultaneously, it proves to be difficult to ascribe these changes to behavioral modulation along the "novelty seeking--anxiety" axis. In mice of other genotypes, changes in the same indices of the open-field behavior were revealed, but these changes were different in their direction. It was suggested that the complex patterns of postnatal behavior was the result of neonatal injections modulating subsequent brain development.     

Interstrain differences in the maturation of congenital reflexes in 101/HY and CBA mice in early postnatal ontogeny]

We have identified interstrain differences in the rate of formation of certain reflexes and parameters of physical development in CBA/LacSto and 101/HY mice. We have shown that in young 101/HY mice, the maturation of reflexes reflecting the development of the vestibular, neurosensory, and neuromuscular systems, such as surface righting, bar holding, negative geotaxis, and auditory startle response, takes place later during postnatal life. Opening of eyes and auditory canals in mice of all studied groups takes place at the same time. In CBA females, maturation of cliff avoidance reflex occurs later than in 101/HY females; hair also appeared later. Body weight of young 101/HY mice of both sexes is significantly higher during the period of postnatal development from day 2 to day 20 than in the CBA strain. However, the relative brain weight was lower in the 101/HY strain. CBA males had a higher brain weight and also showed a faster rate of formation of inborn reflexes. We discuss possible factors underlying the observed difficulties in the rate of formation of reflexes in 101/HY and CBA young mice in relation to general background information about their genetic and neurobehavioral features. The results provide evidence that these differences are genetically determined; the rate of reflex formation does not depend on the overall physical development of mice and is rather due to a delay and/or abnormalities in nervous system development.     

Structural abnormalities of hippocampus in 101/HY mice

We studied cytoarchitectonics of the hippocampus in 101/HY and CBA mice on brain sections stained after Nissl and Timm. In CBA mice, the structure of hippocampus was normal. In 10/HY mice, stratum pyramidale in field CA3 was "splitted" and the density of pyramidal neurons was decreased. Abnormalities were also found in the zone of suprapyramidal projections of mossy fibers (sp-ME), i.e., terminals of axons of the fascia dentata granular cells on the apical dendrites of pyramids. If in CBA mice the sp-MF zone was normal, i.e., looked like a vast compact formation or dense ordered bundle, in 101/HY mice, the sp-MF zone represented a group of scattered, diffuse, and interrupted bundles of varying length, some of which were incorporated in stratum pyramidale. Possible causes of the described morphological abnormalities are discussed, as well as their relation to specific features of biology, behavior, and neurological status of 101/HY mice.     

Behavioral,Neurochemical, and Brain Morphology Features of the 101/HY Mice: A Genetic Model of Some Human Hereditary Diseases

Studies of behavior, neurophysiological reactions, neuromediator synthesis and brain structure of mice of the 101/HY strain (including those of the authors) are reviewed. This mouse strain is characterized by a chromosomal instability because of a recessive mutation mutator-1(mut-1) and the defective DNA excision repair. Experimental studies revealed a number of behavioral and neurological deviations in the 101/HY as compared to the CBA and the C3H mouse strains. These are abnormalities in spatial orientation, altered fear and anxiety reactions, anomalous locomotion, seizure developing in response to agents of various nature, and disturbances of the central nervous system, both structural and biochemical. Genome instability results in a number of neurological mutations, that may lead to the phenotypical effects observed in the 101/HY mice. Since the 101/HY mice partially display signs of severe human hereditary diseases caused by chromosomal instability and defective DNA repair, they can serve as a promising genetic model for these and other diseases related to impairment of the central nervous system.     

Genetic model of some human hereditary diseases: features of behavior, neurochemistry and morphology of the brain in mice of the 101/HY line

Studies of behavior, neurophysiological reactions, neuromediator synthesis and brain structure of mice of the 101/HY strain (including those of the authors) are reviewed. This mouse strain is characterized by a chromosomal instability because of a recessive mutation mutator-1 (mut-1) and the defective DNA excision repair. Experimental studies revealed a number of behavioral and neurological deviations in the 101/HY as compared to the CBA and the C3H strains. These are abnormalities in spatial orientation, altered fear and anxiety reactions, anomalous locomotion, seizure developing in response to agents of various nature, and disturbances of the central nervous system, both structural and biochemical. Genome instability results in a number of neurological mutations, that may lead to the phenotypical effects observed in the 101/HY mice. Since the 101/HY mice partially display signs of severe human hereditary diseases caused by chromosomal instability and defective DNA repair, they can serve as a promising genetic model for these and other diseases related to impairment of the central nervous system.     

Structural Abnormalities of Hippocampus in 101/HY Mice

We studied cytoarchitectonics of the hippocampus in 101/HY and CBA mice on brain sections stained after Nissl and Timm. In CBA mice, the structure of hippocampus was normal. In 101/HY mice, stratum pyramidale in field CA3 was splitted and the density of pyramidal neurons was decreased. Abnormalities were also found in the zone of suprapyramidal projections of mossy fibers (sp-MF), i.e., terminals of axons of the fascia dentata granular cells on the apical dendrites of pyramids. If in CBA mice the sp-MF zone was normal, i.e., looked like a vast compact formation or dense ordered bundle, in 101/HY mice, the sp-MF zone represented a group of scattered, diffuse, and interrupted bundles of varying length, some of which were incorporated in stratum pyramidale. Possible causes of the described morphological abnormalities are discussed, as well as their relation to specific features of biology, behavior, and neurological status of 101/HY mice.     

Modification of the genetic effect of N-nitrosoethylurea in inbred mice by para-aminobenzoic acid

The ability of para-aminobenzoic acid--vitamin (PABA) to influence the sensitivity of mice to alkylating mutagens was studied. PABA had no influence on the cytogenetic effect of thio-TEPA. It was determined that PABA altered the effect of N-ethyl nitrosourea (ENU). The direction of modification depends on animal genotype: pre-treatment with PABA decreases the frequency of chromosome aberrations in bone marrow cells of CBA/LacY and C57BL/JY mice, but significantly increases it in 101/HY mice. The PABA influence on the frequency of gene mutations induced by ENU in melanocytes of mice and revealed by "spot-test" was not determined.     

101/H line mice, a possible model of human chromosomal instability diseases

Chromosome aberrations were studied in cells of embryo liver of 101/H and CBA mice following mutagenic treatment with the alkylating agent--thiophosphamide. Higher sensitivity of chromosomes to aberration induction was found in 101/H mice. After crossing thiophosphamide treated 101/H and CBA males to untreated 101/H and CBA females, the lowest output of dominant lethal mutations was found in the progeny of 101/H females. It is suggested that the 101/H mice are a possible model of inherited diseases with chromosomal instability.     

Preimplantation embryonic mortality--method of studying interstrain differences in the reparative activity of mouse ova

Cytological analysis of preimplantation embryonic death in 101/HY, C57BL/6JY and CBA/lacY females crossed with hybird males F1 (BALB/cYxDBA/2Y) was carried out. Embryonic death was induced by thiophosphamide at a dose of 2 mg/kg, i. p. The maximum preimplantation death was recorded in 101/HY females (38.8%), the minimum in CBA/LacY females (21.9%). In C57BL/6JY females, the maximum preimplantation death accounted for 31.3%. Thus the same chromosome damage induced by thiophosphamide in late spermatids of F1CD2 males caused quantitative differences in embryonic mortality in females of different genotypes. The data obtained evidence that fertilized eggs are capable of repairing part of damage induced by paternal chromosomes. It was demonstrated that the preimplantation embryonic death can be used for studying strain differences from the reparative activity of mouse eggs.     

Studies on the mechanism of transplantation tolerance: I. Do suppressor cells play a role in the induction and maintenance of neonatal transplantation tolerance?

Neonatal transplantation tolerance was induced in CBA (H-2^k) mice to A (H-2^a) mice by injection of (CBA × A)F₁ spleen cells. Animals carrying an A-skin test allograft for more than 4 months without any visible sign of rejection were considered to be permanently tolerant. Permanently tolerant CBA mice were given normal syngeneic spleen cells to abrogate the state of tolerance. Abrogation of tolerance was greatly facilitated by antithymocyte serum (ATS) treatment of tolerant mice prior to the normal syngeneic cell transfer. Survival of A allografts on normal, adult, ATS-treated CBA mice was significantly prolonged (and in many cases “adult” tolerance was achieved) by transfer of spleen cells of syngeneic mice made permanently tolerant at neonatal age. The possible role of the F₁-cell “contamination” in the tolerance-inducing effect of the transferred “tolerant” spleen cells was excluded. The results indicate that ATS-sensitive suppressor cells play a definite role in the induction, maintenance, and transfer of neonatally induced transplantation tolerance.     

Relation of Infection to Tissue Temperature in Mice Infected with Mycobacterium marinum and Mycobacterium leprae

Intravenous and footpad infections with Mycobacterium marinum and footpad infections with M. leprae were compared in the following mouse strains: A/He, BALB/C, CBA, C3H, C57BL, C57L, DBA, 101, and CFW. The results varied a great deal according to mouse strain used. Intravenous injection of high doses of M. marinum resulted in deaths after 28 days of 100% of strain A/He, and none of strain 101; 27 days after injection, the feet and noses of all strain CBA mice, but few of the C57BL, 101, or CFW mice, were involved. Injection of a small dose of M. marinum into the footpad produced visible disease in 5 days in all of the C57BL and 101 mice, but in not more than 60% of the A/He, DBA, and CFW mice; the average amount of swelling at 17 days varied from 4.40 mm in strain C57L to 0.92 in strain 101. After footpad injection of M. leprae, the average plateau harvests varied from 1.3 x 10(7) acid-fast bacteria in strain CBA to 6.5 x 10(5) in strain C57L. The infections in CBA mice extended from the site of inoculation throughout the foot. The temperature was measured rectally, in the footpad, and in the tail. Analysis of all the results revealed little correlation among the three types of infection. There was a strong negative correlation between the tail temperature and the death rate after intravenous injection of M. marinum, and a strong positive correlation between footpad temperature and plateau harvest of M. leprae.     

Ribosomal genes in inbred mouse strains: interstrain and intrastrain variations of copy number and extent of methylation

Quantitative dot hybridization was used to estimate the rDNA copy number in brain tissues of five inbred mouse strains (AKR/JY, NZB/B1OrlY, CBA/CaLacY, 101/HY, and 129/JY), which were obtained from the collection of the Research Center of Biomedical Technologies (Y). In each strain, 9-12 mice aged 1-2 months were examined. The rDNA copy number per diploid genome in strains AKR (range 105-181, mean +/- SD 136 +/- 27) and NZB (129-169, 148 +/- 12) was significantly lower than in strains CBA (172-267, 209 +/- 31), 101 (179-270, 217 +/- 30), and 129 (215-310, 264 +/- 33). Mice of strain NZB were relatively homogeneous in this trait (CV = 8.1%). Strains AKR, CBA, 101, and 129 displayed significant between-group differences, CV varying from 12.5 to 19.9%. The same DNA specimens were digested with MspI or HpaII and used to estimate the extent of methylation of the 28S rDNA region. Regardless of the strain, all mice could be classed into two groups. One group (20 mice) had a methylated fraction accounting for less than 8% of rDNA and included all nine mice of strain NZB, seven out of nine mice of strain 101, and three out of ten mice of strain 129. In the other group (29 mice), the methylated fraction varied from 18 to 38%. A possible role of methylation and the genome dosage of ribosomal genes in phenotypic variation (quantitative trait variation) of inbred mouse strains is discussed.     

Ribosomal genes in inbred mouse strains: Interstrain and intrastrain variation of copy number and extent of methylation

Quantitative dot hybridization was used to estimate the rDNA copy number in brain tissues of five inbred mouse strains (AKR/JY, NZB/B1OrlY, CBA/CaLacY, 101/HY, and 129/JY), which were obtained from the collection of the Research Center of Biomedical Technologies (Y). In each strain, 9–12 mice aged 1–2 months were examined. The rDNA copy number per diploid genome in strains AKR (range 105–181, mean ± SD 136 ± 27) and NZB (129–169, 148 ± 12) was significantly lower than in strains CBA (172–267, 209 ± 31), 101 (179–270, 217 ± 30), and 129 (215–310, 264 ± 33). Mice of strain NZB were relatively homogeneous in this trait (CV = 8.1%). Strains AKR, CBA, 101, and 129 displayed significant between-group differences, CV varying from 12.5 to 19.9%. The same DNA specimens were digested with MspI or HpaII and used to estimate the extent of methylation of the 28S rDNA region. Regardless of the strain, all mice could be classed into two groups. One group (20 mice) had a methylated fraction accounting for less than 8% of rDNA and included all nine mice of strain NZB, seven out of nine mice of strain 101, and three out of ten mice of strain 129. In the other group (29 mice of strains AKR, CBA, 101, and 109), the methylated fraction varied from 18 to 38%. A possible role of methylation and the genome dosage of ribosomal genes in phenotypic variation (quantitative trait variation) of inbred mouse strains is discussed.     

Specific neonatally induced tolerance to Mls locus determinants

Neonatal injection of CBA/HT6T6 (H-2k, Mlsb) mice with adult, Mls-incompatible (CBA/J [H-2k, Mlsd] X CBA/HT6T6)F1 spleen cells results in the abrogation of cell proliferation and interleukin 2 (IL 2) production in bulk mixed lymphocyte cultures, when spleen cells from the inoculated mice are tested at 6 to 8 wk of age with stimulator cells expressing the Mlsd of the tolerizing inoculum. In limiting dilution assays, this tolerant state was manifested in a 25- to 550-fold (280-fold average) decrease in the frequency of precursors of Mlsd-responsive IL 2-producing T cells. Tolerance was specific in that the frequencies of precursors of IL 2-producing cells responding to Con A, allogeneic H-2d, and self-Ia were not affected. The observed low frequency of Mls-responsive cells was due neither to extensive chimerism resulting in the dilution of Mlsd-responsive cells by the nonresponsive F1 cells of the inoculum, nor to the action of suppressor cells. These findings indicate that neonatal injection of Mls-incompatible spleen cells produces a state of specific tolerance by a clonal deletion or inactivation mechanism. This specific tolerance supports the view that 1) the Mls locus encodes or regulates the expression of defined alloantigenic determinants and 2) Mls-incompatible responder mice have specific receptors for Mls determinants on clonally distributed IL 2-producing responder T cells.     

Spontaneous and induced chromosome aberrations in the bone marrow cells of different strains of mice during aging

Sensitivity of bone marrow cell chromosomes to alkylating agent thiophosphamide and to gamma-irradiation has been studied in the course of ageing in 101/H, A/He, CBA, BALB/c and C57BL/6 mice. The effects of both the kinds of mutagenic treatment and of the genotype of the animals on the age-dependent changes in sensitivity of bone marrow cell chromosomes were found. Following gamma-irradiation under our experimental conditions, no variation in the output of chromosomal aberrations was observed between the strains studied. Following thiophosphamide treatment, aged mice of strains 101/H, A/He and CBA showed an increased chromosome instability as compared to young ones. In C57BL/6 mice the level of induced chromosome aberrations was found to be age-independent. Following thiophosphamide treatment, cells with multiple chromosome lesions were found in the bone marrow. The higher instability of aged animals in some strains was mainly due to a sharp increase in the number of such cells. In the intact mice of all the strains studied no age-dependent increase in the number of cells showing structural chromosome aberrations was observed, while accumulation of aneuploid cells varied with genotype.     

An unusual type of locomotion in mice of line 101/Hy]

An unusual type of locomotion--backward movements--was revealed in the 101/HY mouse strain characterized by a chromosomal instability and the defect in DNA repair. The backward movements were found in 60% of mice belonging to the population under study. The manifestation of the character was found to vary: it was high in 16% of the animals. No age and sex differences were found. The backward movements in the 101/HY mouse strain were somewhat similar to the peculiarity in locomotion of the neurological hot-foot mouse mutants. The latter had mild anatomical alterations in the cerebellum. No obvious brain pathology was revealed in the 101/HY mice.     

Molecular manifestations of radiation-induced genome instability: the possibility of chemical modification

The molecular manifestations of radiation-induced genome instability-changes of the DNA structure, the excision DNA repair and the contents of the reactive oxygen forms in bone marrow cells of the repair proficient mice (CBA) and of the repair-defective (101/H) lines in the dynamics up to 185 day after ionizing radiation exposure in the dose of 1.5 Gy were studied. Is was established, that after irradiation in bone marrow cells the descendants with the decreased activity of excision DNA repair and prone to increased changes of DNA structure DHK is arised. The injection of the phenozane in concentrations causing its receptor interaction with cells, did not defend DNA of the bone marrow cells from the radiation injury after the exposure in a sublethal dose, however it exerted influence on long-term changes. Due to the phenosane of the bone marrow cells of the irradiated mice of CBA line exhibited the larger activity in a DNA repair from damages and maintenance of vitality. The bone marrow cells of male mice of repair defective 101/H line, which phenozan was entered before the irradiation, remained unfit to the remuval of DNA damages by the repair, that probably resulted the activations of the program of the maintenance of genome constancy by the apoptosis in the cells--carriers of the structural defects and the cause of animal lethality.     

Mouse Leydig Cells with Different Androgen Production Potential Are Resistant to Estrogenic Stimuli but Responsive to Bisphenol A Which Attenuates Testosterone Metabolism

It is well known that estrogens and estrogen-like endocrine disruptors can suppress steroidogenic gene expression, attenuate androgen production and decrease differentiation of adult Leydig cell lineage. However, there is no information about the possible link between the potency of Leydig cells to produce androgens and their sensitivity to estrogenic stimuli. Thus, the present study explored the relationship between androgen production potential of Leydig cells and their responsiveness to estrogenic compounds. To investigate this relationship we selected mouse genotypes contrasting in sex hormone levels and differing in testosterone/estradiol (T/E₂) ratio. We found that two mouse genotypes, CBA/Lac and C57BL/6j have the highest and the lowest serum T/E₂ ratio associated with increased serum LH level in C57BL/6j compared to CBA/Lac. Analysis of steroidogenic gene expression demonstrated significant upregulation of Cyp19 gene expression but coordinated suppression of LHR, StAR, 3βHSDI and Cyp17a1 in Leydig cells from C57BL/6j that was associated with attenuated androgen production in basal and hCG-stimulated conditions compared to CBA/Lac mice. These genotype-dependent differences in steroidogenesis were not linked to changes in the expression of estrogen receptors ERα and Gpr30, while ERβ expression was attenuated in Leydig cells from C57BL/6j compared to CBA/Lac. No effects of estrogenic agonists on steroidogenesis in Leydig cells from both genotypes were found. In contrast, xenoestrogen bisphenol A significantly potentiated hCG-activated androgen production by Leydig cells from C57BL/6j and CBA/Lac mice by suppressing conversion of testosterone into corresponding metabolite 5α-androstane-3α,17β-diol. All together our data indicate that developing mouse Leydig cells with different androgen production potential are resistant to estrogenic stimuli, while xenoestrogen BPA facilitates hCG-induced steroidogenesis in mouse Leydig cells via attenuation of testosterone metabolism. This cellular event can cause premature maturation of Leydig cells that may create abnormal intratesticular paracrine milieu and disturb proper development of germ cells.     

A factor analysis of behavioral organization in mice]

Laboratory mice with Robertsonian translocation Rb(8,17)1Iem, mice of CBA substrain with the same translocation as well as CBA normal karyotype mice were investigated in several behavioural tests, their results being analysed by means of factor analysis. The genetic differences were revealed in the patterns of factor loadings, in particular in factors "feeding behaviour structure" and "weight loss". The behaviour of mice during logic problem solving (the ability to extrapolate the direction of food stimulus movement was tested) proved to be genotype-dependent in respect to connections with exploratory behaviour indices. Tendencies towards stereotypic or variable (plastic) behaviour patterns were revealed in CBA mice and mice with Rb(8,17)1Iem mice respectively.