Altered laryngeal morphology in Period1 deficient mice.

BACKGROUND: Ultrasonic vocalizations (USV) of mice are produced in and emitted by the larynx. However, which anatomical elements of the mouse larynx are involved and to which aspects of USV they contribute is not clear. Frequency and amplitude parameters of mice, deficient in the clock gene Period1 (mPer1-/- mice) are distinguishably different compared to C3H wildtype (WT) controls. Because structural differences in the larynx may be a reason for the different USV observed, we analyzed laryngeal anatomy of mPer1-/- mice and WT control animals using micro-computed-tomography and stereology. RESULTS: In mPer1-/- mice, we found laryngeal cartilages to be normally arranged, and the thyroid, arytenoid and epiglottal cartilages were similar in diameter and volume measurements, compared to WT mice. However, in the cricoid cartilage, a significant difference in the dorso-ventral diameter and volume was evident. CONCLUSION: Our findings imply that laryngeal morphology is affected by inactivation of the clock gene Period1 in mice, which may contribute to their abnormal USV.

In vivo microscopic voxel-based morphometry with a brain template to characterize strain-specific structures in the mouse brain.

Hundreds of inbred mouse strains are established for use in a broad spectrum of basic research fields, including genetics, neuroscience, immunology, and cancer. Inbred mice exhibit identical intra-strain genetics and divergent inter-strain phenotypes. The cognitive and behavioral divergences must be controlled by the variances of structure and function of their brains; however, the underlying morphological features of strain-to-strain difference remain obscure. Here, in vivo microscopic magnetic resonance imaging was optimized to image the mouse brains by using an isotropic resolution of 80 µm. Next, in vivo templates were created from the data from four major inbred mouse strains (C57Bl/6, BALB/cBy, C3H/He, and DBA/2). A strain-mixed brain template was also created, and the template was then employed to establish automatic voxel-based morphometry (VBM) for the mouse brain. The VBM assessment revealed strain-specific brain morphologies concerning the gray matter volume of the four strains, with a smaller volume in the primary visual cortex for the C3H/He strain, and a smaller volume in the primary auditory cortex and field CA1 of the hippocampus for the DBA/2 strain. These findings would contribute to the basis of for understanding morphological phenotype of the inbred mouse strain and may indicate a relationship between brain morphology and strain-specific cognition and behavior.

The relationship between nephron number, kidney size and body weight in two inbred mouse strains.

While some reports in humans have shown that nephron number is positively correlated with height, body weight or kidney weight, other studies have not reproduced these findings. To understand the impact of genetic and environmental variation on these relationships, we examined whether nephron number correlates with body weight, kidney planar surface area, or kidney weight in two inbred mouse strains with contrasting kidney sizes but no overt renal pathology: C3H/HeJ and C57BL/6J. C3H/HeJ mice had smaller kidneys at birth and larger kidneys by adulthood, however there was no significant difference in nephron number between the two strains. We did observe a correlation between kidney size and body weight at birth and at adulthood for both strains. However, there was no relationship between nephron number and body weight or between nephron number and kidney size. From other studies, it appears that a greater than two-fold variation is required in each of these parameters in order to demonstrate these relationships, suggesting they are highly dependent on scale. Our results are therefore not surprising since there was a less than two-fold variation in each of the parameters examined. In summary, the relationship between nephron number and body or kidney size is most likely to be demonstrated when there is greater phenotypic variation either from genetic and/or environmental factors.

Interstrain differences in susceptibility to non-alcoholic steatohepatitis.

BACKGROUND AND AIM: The pathophysiological mechanisms leading to the development of non-alcoholic steatohepatitis (NASH) remain unclear. There are differences in the susceptibility to NASH between the different species and sexes. The investigation of the precise mechanism of interstrain differences may provide new means by which the pathophysiological mechanisms of NASH may be understood. METHODS: C57BL/6N and C3H/HeN mice were administered a methionine- and choline-deficient (MCD) diet to establish a dietary model of NASH. RESULTS: An elevation of the serum alanine aminotransferase and increased infiltration of inflammatory cells were predominant in C57BL/6N mice at 8 weeks. The increase in the steatosis and lipid contents in the liver was greater in C57BL/6N mice than in C3H/HeN mice. The indices of lipid peroxidation demonstrated by F2-isoprostanes or 8-hydroxy-2'-deoxyguanosine also increased in the livers of C57BL/6N mice. Furthermore, Sirius red staining revealed an increase in the degree of fibrosis in C57BL/6N mice given the MCD diet. As a result, the C57BL/6N strain had a higher susceptibility to NASH than the C3H/HeN mice. The carnitine palmitoyltransferase 1A (in beta-oxidation) mRNA and mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (in ketogenesis) mRNA were downregulated in the C57BL/6N mice in comparison with C3H/HeN mice. There were no differences in the expression of microsomal triglyceride transfer protein or sterol regulatory element binding protein 1 between the C57BL/6N and C3H/HeN mice. CONCLUSION: There were interstrain differences in susceptibility to NASH observed in a rodent dietary model. Further evaluations of the precise molecular mechanism of this interstrain difference may provide some indications of the pathophysiological mechanisms of NASH in humans.

Lower bone cellular activities in male and female mature C3H/HeJ mice are associated with higher bone mass and different pyridinium crosslink profiles compared to C57BL/6J mice.

The female inbred strains of C3H/HeJ (C3H) and C57BL/6J mice (B6), having high and low femoral peak bone mass, respectively, were proposed as models for studying the genetic regulation of bone mass. Here, we compared the known bone phenotype, in 4.5-month-old C3H versus B6 mice, in both genders. Femoral bone mineral content, trabecular bone mass, and thickness at the distal metaphysis were higher in C3H mice. In the long bones, deoxypyridinoline content was lower and pyridinoline/deoxypyridinoline ratios were greater in C3H. Intrafibrillar collagen packing is different not only within strains but also within sexes. Bone resorption activity, evaluated by urinary pyridinium crosslinks and active resorption surfaces in the femoral metaphysis, was lower in C3H. Bone formation activity, evaluated by serum osteocalcin and alkaline phosphatase (ALP) levels, as well as histomorphometric indices of bone formation in the femoral metaphysis and the cortical tibia, was lower in C3H. Conversely, the ALP- and Von Kossa-positive colony-forming units were more numerous in bone marrow cell cultures originating from male C3H. In both strains, resorption and formation activities were lower in males than in females. In C3H, males had lower bone mass than females whereas the opposite was seen in B6. In conclusion, we found that the lower cellular activities in C3H were associated with high cancellous bone mass and pyridinium crosslink levels, which might account for the more mineralized bone in C3H mice compared to that in B6 mice.

Alpha7-nicotinic receptor expression and the anatomical organization of hippocampal interneurons.

C3H and DBA/2 mice differ in their hippocampal inhibitory function, as measured by the inhibitory gating of pyramidal neuron response to repeated auditory stimulation. This functional difference appears to be related to differences in expression of the alpha7 nicotinic cholinergic receptor, which may be generally expressed by interneurons. This study examines the relationship between genetic variation in alpha7 receptor subunit expression and GABAergic interneuron distribution in various regions and layers of the hippocampus in the two mouse strains. Subpopulations of hippocampal interneurons in both mouse strains were found to bind [(125)I]alpha-bungarotoxin. However, the distribution of the [(125)I]alpha-bungarotoxin-positive hippocampal interneurons was significantly different between C3H and DBA/2 mice. In region CA1, and to a lesser extent in region CA3, DBA/2 mice had increased numbers of [(125)I]alpha-bungarotoxin-positive neurons in stratum lacunosum-moleculare and decreased numbers in stratum oriens. Similar differences in GABAergic neuron distribution were observed in region CA1 in the two strains. C3H/DBA/2 F1 animals were backcrossed to the C3H parental strain for six generations, with selection for either the DBA/2 or C3H allelic variant of the alpha7 receptor gene. The distribution of [(125)I]alpha-bungarotoxin labeling closely resembled the DBA/2 parental phenotype in animals retaining the DBA/2 allele of the alpha7 gene. These data suggest that the alpha7 receptor gene locus may influence the anatomical organization of at least a subset of hippocampal interneurons by an as yet unidentified mechanism. This difference in interneuron anatomy may also contribute to functional differences in inhibitory sensory gating between the two strains.

Regional variations in the distribution of small intestinal intraepithelial lymphocytes in three inbred strains of mice.

The regional variation in the intraepithelial lymphocytes (IELs) in the small intestine was examined in BALB/c male and female mice and C3H/He and C57BL/6 male mice. The small intestines were taken from 11 to 12-week-old mice and divided equally into 3 parts (the proximal, middle and distal parts). IELs were isolated from each part of the intestine and analyzed with flow cytometer. The number of IELs was highest in the proximal part and lowest in the distal part. The distribution of IEL subsets was markedly different between the proximal and the distal parts, and that in the middle part showed the intermediate pattern. The percentage of alphabeta T cells were higher in the distal part. In alphabeta T cell subset, the percentage of CD8alphaalpha T cells was higher in the proximal part, whereas those of CD4 and CD4CD8alphaalpha double positive T cells were higher in the distal part. In gammadelta T cell subset, no regional variations were found. The regional variations in the number and subsets of IELs showed almost the same patterns between male and female BALB/c mice and similar patterns among three strains of mice. This strongly suggests that the regional variations in the small intestinal IELs are common to mouse species.

The number of Schmidt-Lanterman incisures is more than doubled in shiverer PNS myelin sheaths.

In the PNS, myelin basic protein (MBP) appears not to be essential for myelination, for in shiverer (shi) and mld mutant mice peripheral nerves, where MBP is not or only poorly expressed, myelination occurs normally. Only a few morphological abnormalities, i.e. reduction in axon calibre and myelin sheath thickness, and aberrant Schwann cell-axon contacts, have been reported. Here, we document a consistent difference between shi and wild type (wt) myelinated sciatic nerve fibres. The number of Schmidt-Lanterman incisures seen in longitudinally and transversely-sectioned sciatic nerves, or in teased fibres stained for the presence of F-actin, is dramatically increased in homozygous shi mice. With both methods, a twofold increase in Schmidt-Lanterman incisure number is seen in 15-day-old mice, the earliest time examined. The increase is slightly greater in nerve fibres from 30- and 90-day-old mice. The overproduction of Schmidt-Lanterman incisures in shi occurs in spite of the fact that the mean diameter of myelinated fibres in shi sciatic nerves is smaller than in wt sciatic nerves. These results lead us to suggest that the increase in Schmidt-Lanterman incisure density in shi compensates for a defect in Schwann cell-axon communication.

Histopathologic observations in weanling B6C3F1 mice and F344/N rats and their adult parental strains.

Weanling Fischer 344/N (F344) rats and the first filial hybrid of C57BL/6 x C3H (B6C3F1) mice and retired breeders from the parental stocks of these strains were monitored over a 5-yr-period by examining the histopathology of selected organs and comparing those results to viral and mycoplasmal serology and the intestinal tract bacterial flora of each animal on an individual basis. Serology gave no evidence of viral infection, but Mycoplasma arthriditis antibodies were detected. Reactivity of serum of adult C57BL/6 female mice with control cells or media (tissue culture, TC) was seen in a significant number of mice. TC reactivity correlated positively with lymphoid perivascular infiltrates, predominantly of the lungs, suggesting an allergic response in development of the lesions. Other lesions of note consisted of Harderian gland inflammation of rats, focal necrotizing lesions of the liver of both species, and thickening of the pleura and adjacent pulmonary interstitium of weanling rats. Embolization of bacteria from the gastrointestinal tract to the liver was considered a possible cause of the liver necrosis in both species. Although lesions of the lung and Harderian gland of the rats are similar to those caused by known viral agents, the cause of the latter could not be determined as these animals were negative for viral antibodies and the former was considered to be related to incomplete pulmonary development in the young rat. Features differentiating the lesions observed in animals of this survey from those caused by viral infection are discussed.

Morphometric studies in inbred and hybrid house mice. VIII. Effects of litter size on brain size and body size.

Litter size, brain size, and body size were examined in inbred and hybrid house mice of three different ages in order to test whether litter size exhibits a positive genetic, but negative environmental association with both brain and body size. As estimated from among-line covariation, litter size showed a positive, but non-significant genetical association with brain and body size. It also showed a significant, negative environmental association with brain and body size, as hypothesized. Over all inbreds and hybrids, litter size explained 8% and 14%, respectively, of the within-strain (environmental) variation in brain and body size. It was concluded that the negative phenotypic association of litter size with brain size and especially body size is the reflection of a well-known negative maternal environmental effect whereby mice with large body sizes tend to produce larger litters of mice with smaller body sizes.

Afferents to the medial nucleus of the trapezoid body and their collateral projections.

Cells and axons that supply direct afferent input to the medial nucleus of the trapezoid body are described. Afferents were intracellularly labeled in brainstem tissue slices of two rodent and two bat species. The main afferents are calyciferous axons from globular bushy cells of the ventral cochlear nucleus. Calyciferous axons were highly consistent across species, projecting directly from the cochlear nucleus, across the midline in the trapezoid body, to the contralateral medial nucleus of the trapezoid body. Within the target nucleus, a typical axon turned sharply away from horizontal to form a large ending, the calyx of Held, around the soma of a single principal cell. Three groups of calyciferous axons were classified based on the path taken from bend to calyx. In subjects younger than four weeks, single axons often formed two calyces, each on a different cell. These calyx pairs were often found on adjacent or vertically aligned cells. In older animals, calyx pairs were more closely aligned, but fewer double calyx axons were seen. A secondary focus of this study was the system of thin collateral branches that characterizes calyciferous axons in all species. The projection patterns of these collaterals suggest that calyciferous axons may provide ascending input to periolivary cell groups with descending projections. In addition to calyciferous afferents, labeled cells that provide input to the medial nucleus of the trapezoid body from adjacent periolivary cell groups are described. Also described is a type of afferent that descends from the level of the lateral lemniscus to the medial nucleus of the trapezoid body.

[The spontaneous aneuploidy of the embryonic fibroblasts in C3H/He and CBA/Ca mouse strains occurring during their neoplastic evolution]. / Spontannaia aneuploidiia émbrional'nykh fibroblastov myshei linii C3H/He i CBA/Ca, voznikaiushchaia v protsesse ikh neoplasticheskoi évoliutsii.

Karyological analysis of 6 cell lines with distinct tumorigenic properties of mouse strains C3H/He and CBA/Ca has been carried out using differential chromosome staining. All the cell lines are characterized by a decreased number of copies of normal chromosome 7, the increased number of normal copies of chromosome 10 being specific of the cell lines with intermediate tumorigenicity. Cell lines with maximum tumorigenicity differed from all other lines by the increased number of copies of chromosome 5 and by the decreased number of copies of chromosome 6. A wide independent variability was observed in the number of chromosomes and of several types of abnormal chromosomes throughout the neoplastic evolution of cells, to begin from the early immortal passages. But the proportion of normal chromosomes per cell in the studied lines revealed relatively stable values. The potential phenotypical heterogenicity of the lines with maximum tumorigenicity, expressed in their clonal progeny, was associated with the instability in the number of chromosome 15 copies in cells of these lines. It is concluded that multiple genetic events are required in the spontaneous neoplastic evolution of fibroblasts, and only specific traits of the karyotypic instability, associated with the variability of the number of copies of specific chromosomes, may constitute the genetic basis for the above process.

[Karyotype characteristics of C3H10T1/2-strain mouse fibroblasts undergoing long-term selection for their capacity to multiply in the presence of ethidium bromide]. / Osobennosti kariotipa fibroblastov myshi linii C3H10T1/2, proshedshikh dlitel'nuiu selektsiiu na sposobnost' razmnozhat'sia v prisutstvii bromistogo étidiia.

Variants Br-0.5 and Br-1 of minimally transformed mouse fibroblasts of C3H10T1/2 line were selected for their ability to proliferate in the medium with 0.5 and 1 mkg/ml of ethidium bromide (EB) toxic for cells of the parent line. Karyological analysis of metaphase chromosomes, stained by Giemsa for G-bands, revealed the number of significant changes in the karyotype of cells resistant to EB. In cells of the resistant sublines the variability of chromosomes was higher than in those of the sensitive population. Two groups of cells are distinguished in the Br-0.5 subline: those with near-diploid and tetraploid chromosome numbers, respectively. The number of polyploid cells in the EB-resistant sublines increases up to 38%, compared to 2% in the parent population. The marker chromosomes in resistant cells originated from translocations, deletions and inversions, with preferential involvement of the material from chromosomes 1.4 and 6. The pericentromeric region of chromosome 4 and the distal region of chromosome I (region 1H1-1H6) were characterized by the increased variability and preferential involvement in rearrangements. In cells of both resistant sublines double mini-chromosomes (1-5 copies per cell) were found. The relation between the revealed chromosomal rearrangements and the mechanism of EB-resistance is discussed.

Hippocampal variation between the inbred mouse strains C3H/HeJ and DBA/2: a quantitative-genetic analysis.

A classical cross-breeding study involving the inbred mouse strains DBA/2 and C3H/HeJ revealed a rather complex mode of inheritance for the following hippocampal variables: size of stratum pyramidale, number of supra-, intra- and infrapyramidal mossy fiber synapses, and the size of terminal fields receiving entorhinal input. A polygenic mode of inheritance was inferred for these phenotypes. For the size of the regio inferior a model containing additive genetic effects only was sufficient to explain the variation between generations. The strain difference may be caused by one genetic factor only. In agreement with previous experiments a strong negative correlation between the number of intra- and infrapyramidal mossy fiber synapses and shuttle-box avoidance performance was found in the genetically heterogeneous F2 population.

Electrophoretic, skeletal, and behavioral divergence of two C3H substrains of mice.

Two C3H substrains of mice, one Australian and one English, were reared under identical conditions in the same laboratory. Electrophoretic and morphometric studies indicated there were genetic differences between the substrains. There were skeletal differences for both metrical and nonmetrical traits of the skeleton scored, and the substrains differed for two of 29 biochemical markers screened electrophoretically. In combination, these results indicate that the two substrains have diverged genetically. A comparison of nest-building behavior indicated that the substrains also differed from one another for the weight of material used to build a nest and the shape of the nest built. The demonstration of genetic divergence in biochemical and morphological traits makes it plausible that these behavioral differences also are due to genetic differences. The implications of these findings for animal studies, especially behavior-genetic studies, are discussed.

Study of mandibular shape in the mouse.

Mandibular shape was compared by the technique of medial axis transformation between four inbred strains of mice fed upon 'hard' and 'soft' diets. Multivariate analysis of component medial axis lengths showed the interstrain contrasts to be slightly greater between animals maintained on 'hard' as opposed to 'soft' diets. The mandibular shape contrasts primarily reflected differences of the ramus compared with the corpus, although the reasons for such changes have yet to be fully investigated.

Cell specific action of 5-thio-D-glucose in mouse testis.

5-thio-D-glucose at 40 mg/kg body weight was administered daily by intraperitoneal injections to male C3H mice. The animals were studied via direct counts of the spermatogenic cells in histologic sections of the testes. These revealed the selective loss or injury of early spermatids followed later in treatment by significantly diminished spermatocyte number. Spermatogonia and Sertoli cells were not significantly affected by thiosugar. After 35 daily doses, testicular and epithelium was noted. The results are indicative of an early drug action on spermatids with later effects on spermatocytes. Recovery began within three weeks of drug withdrawal and was characterized by restoration of the severely disturbed architecture and with the progressive resumption of spermatogenesis. Epididymis sperm counts returned to 91 percent that of control animals; however, testis weights remained somewhat below normal.

Ultrastructure of spontaneous neoplasms induced by diethylnitrosamine and dieldrin in the C3H mouse.

The spontaneous hepatocellular neoplasms of C3H (MTV-ve) male mice were compared with the hepatic tumors induced in these animals by diethylnitrosamine (DEN) and dieldrin. No morphologic differences could be detected by light or electron microscopy between the spontaneous and induced lesions. However, the animals given diethylnitrosamine or dieldrin developed the lesions earlier, in greater numbers and of larger size. The earliest change was the development of foci composed of clear cells. Later nodules appeared which were composed of clear or basophilic cells. These lesions were followed by and presumably progressed to nodules of trabecular hepatocellular carcinomas. It is postulated that in this series, the first morphological step in the neoplastic transformation is the appearance of unusually clear hepatocytes. Ultrastructurally, the clear cells had increased glycogen and lipid droplets and a decrease in smooth endoplasmic reticulum. The basophilic cells seen later resembled the clear cells except for having a greatly increased rough endoplasmic reticulum. Trabecular hepatocellular carcinomas differed from benign nodules in the greater secretory activity of the rough endoplasmic reticulum, in the development of basement membranes at the vascular pole and of microvilli along the lateral cell membranes. The stepwise progression of normal hepatocytes to hepatocellular carcinoma is discussed on the basis of these sequential light microscopic and ultrastructural observations.

Light scattering properties of murine hemopoietic cells.

The light scatter of cells contains information about cell structure and size. Much of this information can be obtained by measuring the light scattered in a flow cytometer in two directions: forward (1.5 degrees-13 degrees) and perpendicular (65 degrees-115 degrees) with respect to the direction of the laser light. For different mouse bone marrow cell types and Sephadex G-25 beads of 10-50 micron diameter, the forward light scatter intensity can be shown to be linearly proportional to the cross-sectional area. The perpendicular light scatter intensity can be shown to depend both on size and degree of structuredness. Therefore, light scatter measurements may be used to obtain overall morphological descriptions of rare cells. By sorting on the basis of light scatter measurements and by subsequent in vivo and in vitro culture assays, it can be shown that the pluripotent hemopoietic stem cell and three committed progenitor cells which represent consecutive stages in the granulocyte/monocyte differentiation series have diameters of 7.1-7.5 micron, and show a complexity of structuredness which increases with differentiation. Since these cells have a low incidence and are only described by their function, such morphological information cannot be obtained by direct microscopic examination of bone marrow. Furthermore, most measurements by flow cytometers can be improved by simultaneous light scatter measurements. Examples are presented which illustrate this in studies of immunofluorescence, leukemic bone marrow, and stem cell purification.