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.

Comparative immunohistochemical study of the dopaminergic systems in two inbred mouse strains (C57BL/6J and DBA/2J).

This study investigated possible neurochemical differences in the brain of two inbred mouse strains, C57BL/6J (C57) and DBA/2J (DBA) that in behavioral, memorization and learning tasks under normal and experimental conditions perform differently or often in an opposite manner. The immunohistochemical study, designed to investigate the dopaminergic system, identified many differences within the midbrain A10 area and less marked differences in areas A9 and A8. The number of dopamine transporter (DAT), vesicular monoamine transporter of type 2 (VMT) and tyrosine hydroxylase (TH) immunoreactive cell bodies was significantly higher in the midbrain of DBA mice than in C57 mice (on average +21.5%, P<0.001 in A10: +9.4% in A9, P<0.05: and +5.9% in A8, P<0.1). The distribution patterns of nerve fibres immunoreactive for same antisera also differed significantly in the two strains, especially at prelimbic, infralimbic and anterior cingulate cortical levels. In C57 mice these fibres were scanty whereas in DBA mice they were well represented. In the nucleus accumbens, also the territorial distribution of DAT immunoreactive nerve fibres differed in the two strains. In the midbrain, the galanin immunoreactive axons were more densely distributed in DBA than in C57 mice whereas neurotensin immunoreactive axons were more densely distributed in C57 than in DBA. These distinct immunohistochemical patterns could help to explain why performance differs in the two mouse strains.

Rodent renal structure differs among species.

In the present study, we histologically and morphometrically investigated species differences in renal structure using laboratory rodents (mice, gerbils, hamsters, rats, and guinea pigs). Morphometric parameters were as follows, 1) diameter of the cortical renal corpuscles, 2) diameter of the juxtamedullary renal corpuscles, 3) percentage of the renal corpuscles with a cuboidal parietal layer, 4) number of nuclei in proximal convoluted tubules (PCTs) per unit area of cortex, 5) semi-quantitative score of the periodic acid-Schiff (PAS) -positive granules in PCTs, and 6) semi-quantitative score of the PAS-positive granules in proximal straight tubules (PSTs). Significant species differences were detected for each parameter, and particularly severe differences were observed in the PAS-positive granules of PCTs and PSTs. Granular scores varied among species and sexes. Vacuolar structures that did not stain with PAS or hematoxylin-eosin were observed in the renal proximal tubules. The appearance and localization of these vacuolar structures differed remarkably between species and sexes.

Structural and functional differences of the carotid body between DBA/2J and A/J strains of mice.

In a previous study, DBA/2J and A/J inbred mice showed extremely different hypoxic ventilatory responses, suggesting variations in their carotid bodies. We have assessed the morphological and functional differences of the carotid bodies in these mice. Histological examination revealed a clearly delineated carotid body only in the DBA/2J mice. Many typical glomus cells and glomeruli appeared in the DBA/2J but not in the A/J mice. The size of the carotid body in the DBA/2J and A/J mice was 6.3 +/- 0.5 x 10(6) and 1.5 +/- 0.3 x 10(6) micro m(3), respectively. The area immunostained for tyrosine hydroxylase, an estimation of the glomus cell quantity, was four times larger in the DBA/2J mice than in the A/J mice. The individual data points in the DBA/2J mice segregated from those in the A/J mice. ACh increased intracellular Ca(2+) in most clusters (81%) of cultured carotid body cells from the DBA/2J mice, but only in 18% of clusters in the A/J mice. These data suggest that genetic determinants account for the strain differences in the structure and function of the carotid body.

Mapping quantitative trait loci that influence femoral cross-sectional area in mice.

Size and shape are critical determinants of the mechanical properties of skeletal elements and can be anticipated to be highly heritable. Moreover, the genes responsible may be independent of those that regulate bone mineral density (BMD). To begin to identify the heritable determinants of skeletal geometry, we have examined femoral cross-sectional area (FCSA) in male and female mice from two inbred strains of mice with divergent FCSA (C57BL/6 [B6] and DBA/2 [D2]), a large genetically heterogeneous population (n = 964) of B6D2F2 mice and 18 BXD recombinant inbred (RI) strains derived from their F2 cross. Femora were harvested from 16-week-old mice and FCSA (bone and marrow space enclosed within the periosteum) was measured at the midshaft by digital image analysis. In all mouse populations examined, FCSA was positively correlated with body weight and weight-corrected FCSA (WC-FCSA) values were normally distributed in the BXD-RI and F2 populations, suggesting polygenic control of this trait. Genome-wide quantitative trait locus (QTL) analysis of the B6D2F2 population revealed regions on four different chromosomes that were very strongly linked to WC-FCSA (chromosomes 6, 8, 10, and X) in both genders. Evidence of gender-specific genetic influences on femoral geometry was also identified at three other chromosomal sites (chromosomes 2, 7, and 12). Supporting evidence for the WC-FCSA QTLs on chromosomes 2, 7, 8, 10, and 12 also was present in the RI strains. Interestingly, none of these WC-FCSA QTLs were identified in our previous QTL analysis of whole body BMD in the same B6D2F2 population. Thus, the genetic determinants of bone size appear to be largely, if not entirely, distinct from those that regulate BMD attainment. The identification of the genes responsible for geometric differences in bone development should reveal fundamentally important processes in the control of skeletal integrity.

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.

Effects of estrous cycle on the mouse kidney morphology.

Although mice kidney morphology shows various sexual dimorphisms, the effect of the estrous cycle has not previously been discussed. In this study, we investigated the effects of the estrous cycle on kidney morphology, including renin-positive areas, of female DBA/2 mice. No effects were confirmed in most of the histometrical parameters, however, the percentage of the renal corpuscles in which cuboidal epithelium covered under 50% of the parietal layer was significantly higher during estrus compared to that during anestrus.

Staining pattern of the brush border and detection of cytoplasmic granules in the uriniferous tubules of female DBA/2Cr mouse kidney: comparison among various fixations and stains.

The proximal straight tubules of the female mouse kidney exhibit heavy periodic acid Schiff (PAS) staining in their brush borders and numerous cytoplasmic granules. In the present study, the female DBA/2Cr mouse kidney was examined, using various fixatives (formalin, PFA, PLP, Zamboni's, Bouin, or Carnoy solution) and various staining methods (HE, PAS, alcian blue, periodic acid methenamine-silver (PAM), toluidine blue, azan, or Congo red). Under azan and PAM, the staining pattern of the brush border was similar to that of PAS, and few effects of the fixative were observed. Cytoplasmic granules were clearly detected with PAM as well as PAS. However, these granules were not detected with Carnoy solution. Furthermore, distribution of granules differed between PAS and PAM.

Contextual-dependent effects of nucleus accumbens lesions on spatial learning in mice.

The effect of nucleus accumbens lesions on radial maze performance of C57BL/6 and DBA/2 mice was assessed under distinct extra-maze cuing conditions. Among sham-lesioned mice, C57BL/6 performed better under rich than poor cuing conditions whereas DBA performed in the same fashion under both conditions. In C57BL/6, a disruptive effect of lesions was found only in mice tested under rich cuing. Conversely, in DBA/2, the lesions improved performance under poor cuing and disrupted performance under rich cuing. In that strain, a possible lesion-induced enhancement of attention to background stimuli improving performance under poor cuing but producing interference under rich cuing is suggested. In general, the lesions effect seemed to depend on the strain predisposition to implement configural or cue-based responding.

A comparison of lung structure in male DBA and C57 black mice and their F1 offspring.

To test whether lung structure in mice is genetically determined, two inbred strains of mice and their F1 offspring of the same age and gender were studied. Body size and lung size were measured, and various morphometric parameters were calculated. Each parameter was analyzed to elucidate the effects of dam, sire, both, or none by a genetic analytical method. The two inbred lines differed in lung and body size as well as in morphometric parameters. These parameters were found to be under genetic control by sire, dam, both, or none. From the data of reciprocal crossing, it appears that there is a maternal effect on the following parameters: body weight, fresh lung weight, fixed lung volume/fresh lung weight, specific lung volume, alveolar wall proportion, mean alveolar wall thickness, and mean linear intercept. Through a more detailed genetic analysis, it was found that specific lung volume in one F1 offspring showed an additive effect. On the other hand, the following parameters indicated heterotic effects (an increase or decrease in dimension compared to the parents with many possibly favorable) in one or both F1 offspring: body weight, fresh lung weight, fixed lung volume/fresh lung weight, specific lung volume, alveolar wall proportion, mean alveolar wall thickness, mean linear intercept, mean chord length of alveoli, mean chord length of alveolar ducts, alveolar surface area, and alveolar surface area to lung volume ratio. These findings indicate that genetic effects are important factors in normal lung development.

A genetic study of neostriatal cholinergic neurones in C57BL/6 and DBA/2 mice.

In the present study Butcher's pharmaco-histochemical technique for acetylcholinesterase has been employed for a morphometrical analysis of striatal cholinergic neurones in crossbred C57BL/6 x DBA/2 F1 mice. The general organization of neostriatal cholinergic systems in hybrid mice was similar to that of parent strains. However, as shown by morphometry, the size of neostriatum in hybrids was larger than that of both parental strains, and the density of striatal cholinergic neurones was significantly lower than that of DBA/2 mice, being close to that of the C57BL/6 strain. The present data indicate that a reduced number of striatal cholinergic neurones is inherited as a dominant trait by these hybrid mice.

A genetic variant in the morphology of the medial preoptic area in mice.

Inbred mice of the DBA/2J and C57BL/6J strains are known to differ in physiological and behavioral characteristics that are partially controlled by nuclei in the preoptic area/anterior hypothalamus. We describe a distinguishing nucleus of darkly staining, densely packed cells, which we term the medioventral pars compacta (MVPC), within the medial preoptic nucleus of DBA/2J, but not C57BL/6J mice. The analysis also indicates that this nucleus is nearly 80% larger in volume in females vs males of the DBA/2J strain. The strain difference may be used to define genetic influences on this neuroanatomical and functional property.

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.

Effect of horse serum on neural cell differentiation in tissue culture.

The effects of various concentrations of horse serum on dissociated mouse glial precursor cells in colony cultures were evaluated. High concentrations (20% or more) favored cell attachment but inhibited cell proliferation and differentiation, whereas lower concentrations (5% to 10%) favored cell proliferation and differentiation. In fetal bovine serum the cells did not attach to culture surfaces to the same degree nor did they achieve the same level of differentiation as in corresponding concentrations of horse serum.

Physical characteristics of mouse sperm nuclei.

The nuclei of epididymal sperm, isolated from C57BL/6J and CBA/J inbred mice by their resistance to trypsin digestion, retain the shape differences of the intact sperm head. Various physical characteristics of these nuclei were measured and compared. The measurement of the projected dimensions of nuclei showed that the CBA nuclei are 13.5% longer than C57BL/6 nuclei (8.64 +/- 0.02 mum compared with 7.61 +/- 0.02 mum), 0.8% narrower (3.51 +/- 0.01 vs. 3.54 +/-0.01 mum) with 6.8% more area (22.34 +/- 0.10 vs. 20.91 +/- 0.09 mum2). However, the volumes of the nuclei as based on reconstructing calibrated electronmicrographs of serial sections of the nuclei indicated that CBA are about 7% smaller than C57BL/6 nuclei (3.72 +/- 0.08 vs. 4.01 +/- 0.03 mum3). The buoyant density of the CBA nuclei is 1.435 +/- 0.002 g/cm3 compared with 1.433 +/- 0.002 g/cm3 for the C57BL/6 nuclei as determined on linear CsCl and Renografin-76 density gradients and confirmed by a technique utilizing physiological tonicities. Therefore, the average mass of the CBA nuclei is less than that of the C57BL/6 nuclei (5.34 +/- 0.12 vs. 5.75 +/- 0.05 pg). The sedimentation velocities at unit gravity of nuclei from 11 inbred strains differ over a range of more than 6% with CBA nuclei sedimenting about 2.0% more slowly than C57BL/6 nuclei. We show that for these nuclei the sedimentation velocity can be related to their buoyant density, volume and a sedimentation shape factor. Within the errors of our measurements of these various characteristics, it was found that C57BL/6 and CBA nuclei have similar sedimentation shape factors. Therefore, the difference in sedimentation velocity between these nuclei appears to be primarily a result of differences in volume. The possible applications of these techniques to the physical separation of sperm are evaluated in the discussion.

Evidence of prolactin cell deficiency in connection with low reproductive efficiency of female 'torpid' mice.

The pituitary of female 'torpid' mice at the age of sexual maturity was deficient in prolactin cells. This deficiency is thought to contribute to the infertility of the affected mice.