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Exposure to environmentally relevant levels of lead (Pb(2+)) during early life produces deficits in hippocampal synaptic plasticity in the form of long-term potentiation (LTP) and spatial learning in young adult rats [Nihei MK, Desmond NL, McGlothan JL, Kuhlmann AC, Guilarte TR (2000) N-methyl-D-aspartate receptor subunit changes are associated with lead-induced deficits of long-term potentiation and spatial learning. Neuroscience 99:233-242; Guilarte TR, Toscano CD, McGlothan JL, Weaver SA (2003) Environmental enrichment reverses cognitive and molecular deficits induced by developmental lead exposure. Ann Neurol 53:50-56]. Other evidence suggests that the performance of rats in the Morris water maze spatial learning tasks is associated with the level of granule cell neurogenesis in the dentate gyrus (DG) [Drapeau E, Mayo W, Aurousseau C, Le Moal M, Piazza P-V, Abrous DN (2003) Spatial memory performance of aged rats in the water maze predicts level of hippocampal neurogenesis. Proc Natl Acad Sci U S A 100:14385-14390]. In this study, we examined whether continuous exposure to environmentally relevant levels of Pb(2+) during early life altered granule cell neurogenesis and morphology in the rat hippocampus. Control and Pb(2+)-exposed rats received bromodeoxyuridine (BrdU) injections (100 mg/kg; i.p.) for five consecutive days starting at postnatal day 45 and were killed either 1 day or 4 weeks after the last injection. The total number of newborn cells in the DG of Pb(2+)-exposed rats was significantly decreased (13%; P<0.001) 1 day after BrdU injections relative to controls. Further, the survival of newborn cells in Pb(2+)-exposed rats was significantly decreased by 22.7% (P<0.001) relative to control animals. Co-localization of BrdU with neuronal or astrocytic markers did not reveal a significant effect of Pb(2+) exposure on cellular fate. In Pb(2+)-exposed rats, immature granule cells immunolabeled with doublecortin (DCX) displayed aberrant dendritic morphology. That is, the overall length-density of the DCX-positive apical dendrites in the outer portion of the DG molecular layer was significantly reduced up to 36% in the suprapyramidal blade only. We also found that the area of Timm's-positive staining representative of the mossy fibers terminal fields in the CA3 stratum oriens (SO) was reduced by 26% in Pb(2+)-exposed rats. These findings demonstrate that exposure to environmentally relevant levels of Pb(2+) during early life alters granule cell neurogenesis and morphology in the rat hippocampus. They provide a cellular and morphological basis for the deficits in synaptic plasticity and spatial learning documented in Pb(2+)-exposed animals.

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Estrogens are known to have broad effects on neuronal plasticity, but their specific role in neuronal cell death has not been determined. In the present study, we investigated the effects of beta-estradiol on an experimental model of apoptosis of granule cells of the dentate gyrus, i.e., apoptosis induced by intraventricular injection of the microtubule polymerization inhibitor colchicine. Cell death was characterized with multiple methods, including TUNEL and DNA electrophoresis. Nonrandom digestion of DNA was observed within 8-10 hours after colchicine injection, followed by condensation and fragmentation of granule cell nuclei and extensive anterograde degeneration of mossy fibers/terminals in 2 days. We compared the outcomes of the above-described manipulation in ovariectomized or sham-operated rats and animals treated daily with beta-estradiol or vehicle. Animals were lesioned with colchicine or vehicle 2 weeks after ovariectomy or sham operation. Beta-estradiol or vehicle was administered for 1 week prior to lesion and was continued for a further 2 weeks. Total numbers and densities of granule cells in different animal groups were counted by stereology in various anteroposterior levels of the hippocampus. Our results show that ovariectomy intensifies colchicine-induced granule cell apoptosis, which is ameliorated by exogenous beta-estradiol. In doses that ameliorate the adverse effect of ovariectomy, exogenous beta-estradiol appears to have no effect of preventing granule cell death in animals with intact ovaries; i.e., an estrogen excess is not more neuroprotective than physiological levels of these hormones. Taken together, our results indicate that estrogen deprivation increases the vulnerability of hippocampal neurons to injury and may predispose to neurological diseases occurring after menopause.

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We quantified the amount of amyloid beta-peptide (Abeta) immunoreactivity as well as amyloid deposits in a large cohort of transgenic mice overexpressing the V717F human amyloid precursor protein (APP(V717F+/-) TG mice) with no, one, or two mouse apolipoprotein E (Apoe) alleles at various ages. Remarkably, no amyloid deposits were found in any brain region of APP(V717F+/-) Apoe(-/-) TG mice as old as 22 mo of age, whereas age-matched APP(V717F +/-) Apoe(+/-) and Apoe(+/+) TG mice display abundant amyloid deposition. The amount of Abeta immunoreactivity in the hippocampus was also markedly reduced in an Apoe gene dose-dependent manner (Apoe(+/+) > Apoe(+/-) >> Apoe(-/-)), and no Abeta immunoreactivity was detected in the cerebral cortex of APP(V717F+/-) Apoe(-/-) TG mice at any of the time points examined. The absence of apolipoprotein E protein (apoE) dramatically reduced the amount of both Abeta(1-40) and Abeta(1-42) immunoreactive deposits as well as the resulting astrogliosis and microgliosis normally observed in APP(V717F) TG mice. ApoE immunoreactivity was detected in a subset of Abeta immunoreactive deposits and in virtually all thioflavine-S-fluorescent amyloid deposits. Because the absence of apoE alters neither the transcription or translation of the APP(V717F) transgene nor its processing to Abeta peptide(s), we postulate that apoE promotes both the deposition and fibrillization of Abeta, ultimately affecting clearance of protease-resistant Abeta/apoE aggregates. ApoE appears to play an essential role in amyloid deposition in brain, one of the neuropathological hallmarks of Alzheimer's disease.

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The weaver (wv) mutant mouse manifests severe locomotor defects, a deficiency in granule cells of the cerebellum, and cellular deficits in the midbrain dopaminergic system. The wv phenotype is associated with a missense mutation in the pore region of the G-protein-gated inwardly rectifying potassium channel, GIRK2. The homozygous male wv mouse is essentially infertile due to an inadequate level of sperm production. Females are fertile although they also manifest the neurological phenotype. Homozygotes of both sexes have reduced body weight. We have evaluated the hypothalamic-pituitary-gonadal axis in heterozygote and homozygote male and female wv mutants in comparison with wild-type controls. Testicular weight was significantly reduced in the homozygous males, due to degenerative changes of seminiferous epithelium. Serum and pituitary content of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin were normal in all groups, and the normal sex differences were noted (FSH and LH higher in males, prolactin higher in females). Pituitary growth hormone (GH) concentration was normal, with control and mutant males showing higher GH than females. Serum testosterone levels were normal in the mutants, as was testicular testosterone. Testicular alpha-inhibin content was mildly reduced, but high in proportion to testicular weight. The defect in spermatogenesis appeared predominantly in the postmeiotic stages. In situ hybridization was consistent with expression of some GIRK2 mRNA isoforms in seminiferous epithelium. There were no significant differences between genotypes in the levels of dopamine, dihydroxyphenylacetic acid, serotonin and 5-hydroxyindoleacetic acid in the mediobasal and preoptic hypothalamic regions. Homovanillic acid levels in these two areas were, however, reduced in wv homozygotes compared to wild-type animals. In the light of normal pituitary hormone levels, normal hypothalamic monoamine concentrations and normal sex differences in gonadotropins, we conclude that the infertility in the male homozygote wv mouse lies within the tubule and is probably a primary defect in the germ cells. The hormonal data suggest that Leydig cell function, and at least some aspects of Sertoli cell function, are normal in the mutant mice.

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A mutation in the gene Girk2 that encodes an inwardly rectifying potassium channel is the genetic defect causing the behavioral and pathologic abnormalities of the weaver mutant mouse. Of the pathologic abnormalities, the best studied is the neuronal degeneration that occurs in the cerebellar cortex and in the midbrain dopaminergic neurons. A detailed characterization of the topographic and temporal expression of Girk2 is fundamental to elucidate the mechanisms underlying neurodegeneration in these mutant mice. In this study we utilized in situ hybridization to determine the expression of Girk2 mRNA during prenatal and postnatal development in the murine central nervous system (CNS). Girk2 expression was seen in multiple regions of embryonic CNS including the cerebellum and midbrain. During postnatal development, the highest expression was seen in the cerebellum, midbrain and hippocampus. However, since the developing cerebellum undergoes significant neuronal loss due to the degeneration of granule cell precursors, Girk2 mRNA expression in this area decreases progressively.

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The phenotypic effect of the weaver mutation in the ventral midbrain of homozygous mutants is associated with the progressive loss of dopaminergic neurons. To discover whether the number of mesencephalic dopaminergic cells is altered in weaver heterozygotes (wv/+), we studied mice between 20 and 365 days of age. We counted tyrosine hydroxylase (TH)-immunopositive cells in the substantia nigra (SN), retrorubral nucleus (RRN), and ventral tegmental area (VTA), and measured cross-sectional areas of neuronal somata in the SN of wv/+ and age-matched wild-type controls (+/+). The number of TH-positive cells in the wv/+ ventral midbrain was on average 13% lower than normal. Cell loss was detected selectively in the SN (12%) and VTA (23%). The areas of somatic profiles in the wv/+ nigral neurons were on average reduced by 9.8%. The neuronal losses in the SN and VTA correlated with a 13.8% reduction in dopamine level in the ventral striatum in wv/+ mice at 14-16 months of age. Our findings imply that a single dose of the weaver gene in the mouse is associated with cellular damage leading to a chronic deficiency in the mesostriatal dopaminergic system.

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Degeneration of neurons in two structures, the cerebellum and the dopaminergic neurons in the ventral midbrain, is a well characterized action of the weaver gene. In order to see whether the gene has effects prenatally, both the cerebellum and the ventral midbrain were examined in mouse embryos genotyped for the weaver gene (wv, Girk2) on day E19. Anatomically matched sections of the midline cerebellar vermis were quantitatively analyzed 2 h after the dams were given a single injection of [3H]thymidine. A gene-dose effect was seen in the retardation of fissure development. This was more pronounced in homozygotes (wv/wv) and less so in heterozygotes (wv/+) when compared with wild type controls (+/+). Quantitative measures of the following features showed stepwise differences between genotypes so that the wv/wv are most affected and wv/+ are somewhat affected compared with +/+: surface length of the midline vermis, area of the entire midline vermis and the external germinal layer (egl), total number of cells in the egl, [3H]thymidine-labeled and -unlabeled egl cells, cells in the Purkinje cell layer, cells in the region of the deep nuclei, [3H]thymidine-labeled cells in the Purkinje cell layer (presumptive proliferating Bergmann glia), and [3H]thymidine-labeled cells in the region of the deep nuclei. In contrast to the obvious phenotypic effects of wv in the embryonic cerebellum, qualitative immunocytochemical examination of tyrosine hydroxylase staining in the ventral midbrains of the same embryos showed that the position and density of the presumptive dopaminergic neurons was similar in all genotypes.

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In homozygous weaver (wv/wv) mutant mice, nearly 50% of the dopaminergic substantia nigra neurons degenerate by postnatal day 20. We have now determined that the total number of dopaminergic neurons in the ventral midbrains of a litter of obligatory homozygous weaver pups and a litter of normal wild-type control pups indicates that no significant differences are present between groups at birth. To test the hypothesis that the subsequent degeneration of these neurons is linked to their time of origin, [3H]thymidine autoradiography was combined with tyrosine hydroxylase immunocytochemistry to construct neurogenetic timetables on postnatal day 20 in wild-type mice and weaver homozygotes. Both groups have the same span of neurogenesis but have statistically different proportions of neurons generated on specific days. In wild-type mice, more than half of the dopaminergic neurons originate on or after embryonic day 12. In contrast, over two-thirds of the surviving dopaminergic neurons in homozygous weaver mice originate on or before embryonic day 11. Our data suggest that the weaver gene does not interfere with the generation of dopaminergic neurons, but it preferentially kills late-generated dopaminergic neurons between birth and postnatal day 20.

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In the neurological mutant mouse weaver (wv/wv), the majority of males are infertile due to hypospermatogenesis. Heterozygous weaver mice (wv/+) cease mating successfully when males reach an average age of 3.5 months. The contents of epididymal fluid were scored for the number of sperm and sperm motility in wv/wv, wv/+ and controls. Testes were examined in mice of the three genotypes at various ages using light and electron microscopy. In wv/+ males, sperm counts were significantly lower than in controls and were significantly higher than in wv/wv. The seminiferous epithelium in weaver mice appears depleted soon after puberty and a wide range of degenerative changes was identified in both germ cells and Sertoli cells. Analogous cellular aberrations were detected in heterozygous males, but they appeared at an older age and were not as severe as in wv/wv. We hypothesize that in weaver homo- and heterozygosity the damage of Sertoli cells may induce degeneration of germinal cells and particularly affect the most advanced spermatogenic cells.

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The pathologic phenotype of the testis in both prepuberal and postpuberal male weaver mutant mice was studied by light microscopy. Morphometric analysis of seminiferous tubules was carried out. Epididymal fluid was examined for the presence of spermatozoa. The seminiferous tubules of 21-day-old prepuberal weaver mutant mice lacked patent lumina and had more degenerated cells than control mice. Fifty-six day-old weaver mutants had many germinal epithelial cells located within the adluminal compartment that were in advanced stages of degeneration. Round spermatids were enlarged and multinucleated. Round spermatids and spermatocytes had sloughed into the lumen. Compared to control mice, elongated spermatids were seen less frequently. In older weaver mice, the degenerative process involved germ cells in both the adluminal and basal compartments. In 143- and 226- day-old weaver mutants, the Sertoli cells were atrophic. Diameters of seminiferous tubules in weaver mice were significantly reduced when compared to control mice. Sperm were either absent or very low in number in the epididymal fluid of postpuberal weaver mice. We conclude that spermatogenesis is abnormal in male weaver mutant mice. The testicular phenotype is characterized by a degenerative process that affects both germ cells and supporting cells.

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The effect of colchicine and cytochalasin B and D on the process of glucose and plant oil absorption in the small intestine of rats was studied using the light and electron microscopy and biochemical methods. The colchicine and CB, CD action on the elements of enterocytes' apical contractile complex and cytoskeleton inhibited the absorption thus suggesting the major role of endocytosis in the process of nutrients absorption in the small intestine.

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Polyethylene glycol (PEG)-4000 is proposed as a tracer of intestinal macromolecular permeability. The reproducibility of permeability testing with PEG-4000 and the mechanism of its penetration through intestinal mucosa were studied in adult rats. Permeability measurement for PEG-4000 was reproducible when repeated twice for 2 days. This makes it possible to repeat PEG-4000 permeability testing before and after any experimental impact on the intestine. I.p. administration of colchicine to rats (125 micrograms/100 g b. w.) significantly inhibited intestinal absorption of PEG-4000 fed to the animals 3 hours later. Hence, PEG-4000 penetration through the intestinal mucosa is mediated by the system of enterocyte cytoplasmic microtubules. Mucosal permeability for PEG-4000 may be consequently considered as a valuable model of permeability for protein macromolecules.

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