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This study describes the topography, borders and divisions of the globus pallidus in the Brazilian short-tailed opossum (Monodelphis domestica) and distribution of the three calcium binding proteins, parvalbumin (PV), calbindin D-28k (CB) and calretinin (CR) in that nucleus. The globus pallidus of the opossum consists of medial and lateral parts that are visible with Nissl or Timm's staining and also in PV and CR immunostained sections. Neurons of the globus pallidus expressing these proteins were classified into three types on the basis of size and shape of their soma and dendritic tree. Type 1 neurons had medium-sized fusiform soma with dendrites sprouting from the opposite poles. Neurons of the type 2 had medium-to-large, multipolar soma with scarce, thin dendrites. Cell bodies of type 3 neurons were small and either ovoid or round. Immunostaining showed that the most numerous were neurons expressing PV that belonged to all three types. Density of the PV-immunopositive fibers and puncta correlated with the density of the PV-labeled neurons. Labeling for CB resulted mainly in the light staining of neuropil in both parts of the nucleus, while the CB-expressing cells (mainly of the type 2) were scarce and placed only along the border of the globus pallidus and putamen. Staining for calretinin resulted in labeling almost exclusively the immunoreactive puncta and fibers that were distributed with medium-to-high density throughout the nucleus. Close to the border of globus pallidus with the putamen these fibers (probably dendrites) were long, thin and varicous, while more medially bundles of thick, short and smooth fibers predominated. Single CR-ir neurons (all of the type 3) were scattered through the globus pallidus. Colocalization of two calcium binding proteins in one neuron was. never observed. The CB-ir puncta (probably terminals of axons projecting to the nucleus) frequently formed basket-like structures around the PV-ir neurons. Therefore, the globus pallidus in the opossum, much as that in the rat, consists of a heterogeneous population of neurons, probably playing diversified functions.

A comparative quantitative study of the somatosensory thalamocortical connections in the rat and rabbit, labeled with the fluorescent retrograde tracer Fluoro-Gold (FG), was conducted by means of unbiased stereology. FG was injected into the primary somatosensory cortex of the rat and rabbit in different age groups from P0 to P180 (P-postnatal day). The numerical density of retrogradely labeled the ventroposterolateral (VPL) projection neurons was analyzed. A significant decrease in this parameter was observed during the first two weeks of postnatal life in both studied species. Changes of the neuropil volume and selective elimination of early cortical connections stemming from the VPL may possibly cause this process. A withdrawal of axon collaterals from the expanded cortical sites as well as apoptosis (existing both in the VPL and parietal cortex) contribute to a decrease in the numerical density. Our observations allow us to conclude that the thalamocortical somatosensory connections established before the birth undergo significant quantitative changes in both studied species during the first two weeks of postnatal life and this period seems to be crucial for maturation of the thalamocortical loop.

Distribution of mitochondria as well as other intracellular organelles in mammalian cells is regulated by interphase microtubules. Here, we demonstrate a role of microtubules in the mitochondrial biogenesis using various microtubule-active drugs and human osteosarcoma cell line 143B cells and rat liver-derived RL-34 cells. Depolymerization of microtubules by nocodazole or colchicine, as well as 2-methoxyestradiol, a natural estrogen metabolite, arrested asynchronously cultured cells in G(2)/M phase of cell cycle and at the same time inhibited the mitochondrial mass increase and mtDNA replication. These drugs also inhibited the mitochondrial mass increase in the cells that were synchronized in cell cycle, which should occur during G(1) to G(2) phase progression in normal conditions. However, stabilization of microtubules by taxol did not affect the proliferation of mitochondria during the cell cycle, yet a prolonged incubation of cells with taxol induced an abnormal accumulation of mitochondria in cells arrested in G(2)/M phase of cell cycle. Taxol-induced accumulation of mitochondria was not only demonstrated by mitochondria-specific fluorescent dyes but also evidenced by the examination of cells transfected with yellow fluorescent protein fused with mitochondrial targeting sequence from subunit VIII of human cytochrome c oxidase (pEYFP) and by enhanced mtDNA replication. Two subpopulations of mitochondria were detected in taxol-treated cells: mitochondria with high Delta(psi)(m), detectable either by Mito Tracker Red CMXRos or by Green FM, and those with low Delta(psi)(m), detectable only by Green FM. However, taxol-induced increases in the mitochondrial mass and in the level of acetylated (alpha)-tubulin were abrogated by a co-treatment with taxol and nocodazole or taxol and colchicine. These data strongly suggest that interphase microtubules may be essential for the regulation of mitochondrial biogenesis in mammalian cells.

There is general agreement that oxidative stress may induce apoptotic and necrotic cell death. Recently it has been shown that NADH can be considered an important antioxidant as it reacts with peroxyl and alkoxyl radicals under in vitro conditions. Therefore, in the present study we hypothesized that an increase in intracellular NADH using specific substrates will protect RL-34 cells against cytotoxicity of 2'-azobis (2-amidinopropane) dihydrochloride (AAPH), which is a peroxyl radical generating compound. Cells treated for 24 hours with 6.0 mM AAPH were severely damaged: mitochondria were vacuolated, and the level of free radicals significantly increased. Both apoptotic and necrotic cells were detected (11.1% and 11.4%, respectively) even after 5 hours of treatment. Pretreatment of the cells with substrates which increase the intracellular level of NADH, such as lactate, beta-hydroxybutyrate, and ethanol, distinctly inhibited AAPH-induced reactive oxygen species (ROS) formation and cell death. On the other hand, acetoacetate (AcA), which decrease the intracellular level of NADH, had opposite effects. Interestingly, NADH-generating substrates augment, while AcA reduced superoxide radical formation induced by AAPH. These results may suggest that although NADH generating substrates may exert some deleterious effects within a cell by inducing reductive stress, they diminish alkoxyl or peroxyl radical cytotoxicity. The protection is associated with a decrease in ROS formation measured by dichlorofluorescein, but with an increase in superoxide radical formation.