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.     

Strain-specific response in mice to the neonatal administration of ACTH(4-10) fragment: behavior, neurochemistry, and brain morphology

Neonatal injection of the ACTH4-10 fragment (5 micrograms daily for five days) caused genotype-dependent changes in concentrations of some monoaminergic neuromediators and their metabolites in hippocampus and brain stem of adult CBA and 101/HY mice. The catecholaminergic neurons increased in number in hypothalamic zona incerta of adult 101/HY mice. Neonatal injection of the peptide caused also genotype-dependent changes in the exploratory behavior of adult animals. Sound sensitivity was reduced in the 101/HY mice, whereas no sensitivity was revealed in both control and experimental groups of the CBA mice. The effects discovered were suggested to be caused by changes in neuronal differentiation.     

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.     

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.     

TOPOGRAPHICAL AND SUBCELLULAR LOCALIZATION OF CHOLINE ACETYLTRANSFERASE IN RAT HIPPOCAMPAL REGION

—Choline acetyltransferase (ChAc) was localized in discrete layers in hippocampus regio superior and in area dentata. The highest activity in hippocampus was found in a narrow infrapyramidal zone, but high activities were also observed in the rest of stratum oriens and in stratum pyramidale. In area dentata the highest activities were found in a narrow supragranular zone and in hilus fasciae dentatae. The localization corresponded very closely to that of acetylcholinesterase. The main part of ChAc activity was confined to the synaptosome fraction. The results are compatible with the view that pyramidal and granular cells are excited by cholinergic boutons, mainly located on the basal or apical dendrites near the somata.     

Immediate effects of maternal separation on the development of interneurons derived from medial ganglionic eminence in the neonatal mouse hippocampus

Aversive experiences, including maternal separation (MS), have been known as a risk for abnormal hippocampus development. Given that impairment of GABA inhibitory system is known as one of the common features of the abnormal hippocampal development induced by MS, we examined whether the MS on 4‐day‐old (P4) mice for 24 hr abolishes the interneuron development. We observed that the MS reduced the volume of dorsal hippocampus on P14 as long‐term effects. In addition, the MS decreased the number of parvalbumin (PV)‐positive interneuron on P14 and P28 in the dorsal hippocampus. We further examined the immediate effects of MS by measuring the percentage of glutamic acid decarboxylase (GAD) 67‐positive interneurons among the immature interneurons derived from medial ganglionic eminence (MGE) progenitors marked in nkx2.1cre;β‐geo EGFP mice. During normal development from P4 to P5, the percentage of GAD67‐positive interneurons among the MGE‐derived interneurons in the dorsal hippocampus was significantly increased from 42.29% to 70.73% in the stratum pyramidale of the CA1 and increased from 46.4% to 56.99% in the stratum pyramidale of the CA2/3 region. However, the increase was not observed on P5 among the mice treated with the MS. These results suggest that the maturation of interneurons was suppressed by the MS. The suppressed maturation of interneurons may be one of the causes of the reduced volume of the hippocampus and PV+ interneurons observed in the hippocampus on P14 and P28 as a consequence of the MS during neonatal stage.     

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.     

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.     

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.     

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.     

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.     

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.     

Phosphorylated extracellular signal-regulated kinase 1/2 immunoreactivity and its protein levels in the gerbil hippocampus during normal aging

Phosphorylated extracellular signal-regulated kinase (pERK) mediates neuronal synaptic plasticity, long-term potentiation, and learning and memory in the hippocampus. In this study, we examined pERK1/2 immunoreactivity and its protein level in the gerbil hippocampus at various ages. In the postnatal month 1 (PM 1) group, very weak pERK1/2 immunoreactivity was detected in the hippocampus. In the CA1 region, pERK1/2 immunoreactivity was considerably increased in the stratum pyramidale in the PM 6 group. Thereafter, pERK1/2 immunoreactivity was decreased. In the CA2/3 region, pERK1/2 immunoreactivity increased in an age-dependent manner until PM 12. Thereafter, numbers of pERK1/2-immunoreactive neurons were decreased. However, in the mossy fiber zone, pERK1/2 immunostaining became stronger with age. In the dentate gyrus, a few pERK1/2-immunoreactive cells were observed until PM 12. In the PM 18 and 24 groups, numbers of pERK1/2-immunoreactive cells were increased, especially in the polymorphic layer. In Western blot analysis, pERK1/2 level in the gerbil hippocampus was increased with age. These results indicate that total pERK1/2 levels are increased in the hippocampus with age. However pERK1/2 immunoreactivity in subregions of the gerbil hippocampus was changed with different pattern during normal aging.     

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.     

The 1×C3 panel of recombinant inbred mouse strains. Presence or absence of pathologic neurologic traits of one of parental strains

Mice from the earlier developed recombinant inbred strains (RIS), which were derived by crossing 101/HY mice (carrying the mut-1 allele determining increased susceptibility to the mutagenic action of alkylating compounds) with C3H/Sn mice (lacking this trait), were tested for the presence of two neurological pathologies, audiogenic epilepsy and splitting of pyramidal cell layer of the CA3 hippocampal field (specific only to 101/HY mice). It was demonstrated that segregation of RIS relative to these traits was independent from the presence or absence of the mut-1 allele. These findings suggested the appearance of mut-1-independent mutations in the 101/HY mice, which resulted in the development of neurological pathologies. The appearance of such mutations can be the consequence of the genetic repair defects, earlier observed in the mice with this genotype.     

SP-SR interneurones: a novel class of neurones of the CA2 region of the hippocampus

The CA2 region of the hippocampus has distinctive properties and inputs and may be linked with the pathology of specific psychiatric and neurological disorders. It is, therefore, important to understand CA2 circuitry and its involvement in the circuitry of the hippocampus. Properties of CA2 basket cells have been reported. However, other classes of CA2 interneurones with cell bodies located in stratum pyramidale remained to be described. In this study, the unusual axonal arbors of a novel subclass of dendrite-preferring CA2 interneurones whose somata are located in the pyramidal cell layer was revealed following intracellular recordings and biocytin labeling. One to four apical dendrites emerged from the soma, branched in stratum radiatum (SR) forming a tuft, but rarely penetrated stratum lacunosum-moleculare (SLM). One or two basal dendrites branched close to the soma, the branches extended through stratum oriens (SO) and often reached the alveus. Unlike CA2 bistratified cells, the axons of these cells arborized almost exclusively in SR with few, if any, branches extending to stratum pyramidale (SP), SO, or SLM. These interneurones again, unlike bistratified cells, were immunonegative for parvalbumin and cholecystokinin. Electrophysiologically, they were similar to some CA2 basket and bistratified cells in that they presented a "sag" in response to hyperpolarizing current injections and displayed spike frequency adaptation. They targeted the apical dendrites of neighboring CA2 pyramidal cells and received inputs from them.     

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.     

Altered production and renewal of natural killer cells in B-lymphocyte-deficient CBA/N mice

The present study was designed to measure by quantitative and kinetic methods the production and renewal of natural killer (NK) cells in congenitally B-lymphocyte-deficient (CBA/N) mice. The total NK activity (percent specific lysis corrected for changes in whole organ cellularity) of the bone marrow and spleen of immunologically normal (CBA/CaJ) and CBA/N mice was assayed prior to and immediately after 48 h treatment (2 X/day, i.p.) with the cell cycle poison hydroxyurea (HU) and at various intervals throughout the subsequent post-HU recovery period. The total NK activity (TNKA) of untreated CBA/N bone marrow was 154% of that of CBA/CaJ bone marrow while the TNKA of CBA/N spleen was not significantly different (112%) from that of CBA/CaJ spleen. At the conclusion of 48 h HU, bone marrow TNKA of CBA/N and CBA/CaJ mice fell to 60 and 49%, respectively, of their saline-injected (2 X/day, i.p.) control levels, while spleen TNKA fell to 42 and 61%, respectively, of their saline-injected control levels. In the bone marrow, NK cell depletion in response to HU was more rapid in CBA/N mice (day 0.5 after HU) than in CBA/CaJ mice (day 2 after HU). TNKA of the spleen also decreased more rapidly in CBA/N mice (day 2 after HU) than in CBA/CaJ mice (day 3 after HU). The data indicate an enhanced production and turnover of NK cells in CBA/N mice relative to CBA/CaJ mice. Moreover, increased production and renewal of NK cells in CBA/N mice together with virtually unchanged levels of NK activity (112% of CBA/CaJ mice) in CBA/N mouse spleens indicate that mature lytic NK cells in CBA/N spleen but not bone marrow have a significantly shorter post-mitotic life span than do NK cells in the spleens of immunologically normal (CBA/CaJ) mice.     

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.     

Reversal of sexual dimorphism in splenic T lymphocyte responses after trauma-hemorrhage with aging

Previous studies have demonstrated that hemorrhagic shockproduces immunodepression in young male mice, whereas theimmunoresponsivness in young proestrus female mice is enhanced undersuch conditions. This sexually dimorphic immune response to hemorrhageappears to be related to high estrogen and testosterone levels infemales and males, respectively. Nonetheless, it is unknown what impact the age-related decline in the sex steroid levels has on the immune response after hemorrhage. To study this, young (2-3 mo) and aged (18-19 mo) male and female CBA/J NIA mice were subjected tolaparotomy (i.e., soft tissue trauma) and hemorrhage (35 ± 5 mmHg for90 min and fluid resuscitation) or sham operation. Twenty-four hours later, splenocyte responses were assessed in vitro. Splenic T lymphocyte responses [i.e., proliferation, interleukin-2 (IL-2) and interferon- (IFN-) release] were depressed in youngmales and enhanced in young females after trauma-hemorrhage. Incontrast, in the aged male and female groups these parameters ofsplenocyte function were reversed after trauma-hemorrhage (i.e.,increased proliferation and IL-2 release in aged males compared withsuppressed proliferation and IFN- release in aged females).Furthermore, the release of the immunosuppressive cytokine IL-10inversely correlated with the age- and gender-related changes insplenocyte responses after trauma-hemorrhage. Thus the sexuallydimorphic immune response in young males and females totrauma-hemorrhage appears to reverse as sex hormone levels decline with age.