Cell Type-Specific Properties of Subicular GABAergic Currents Shape Hippocampal Output Firing Mode

GABAergic function of the subiculum is central to the regulation of hippocampal output activity. Subicular neuronal networks are indeed under potent control by local inhibition. However, information about the properties of GABAergic currents generated by neurons of this parahippocampal area in normal tissue is still missing. Here, we describe GABA_A receptor (GABA_AR)-mediated phasic and tonic currents generated by principal cells (PCs) and interneurons (INs) of the rat subiculum. We show that in spite of similar synaptic current densities, INs generate spontaneous IPSCs (sIPSCs) that occur less frequently and exhibit smaller charge transfer, thus receiving less synaptic total current than PCs. Further distinction of PCs between intrinsically bursting (IB) and regular-spiking (RS) neurons suggested that sIPSCs generated by the two PC sub-types are likely to be similar. PCs and INs are also controlled by a similar tonic inhibition. However, whereas a comparable tonic current density is found in RS cells and INs, IB neurons are constrained by a greater inhibitory tone. Finally, pharmacological blockade of GABA_AR did not promote functional switch of RS neurons to IB mode, but influenced the bursting propensity of IB cells and released fast spiking activity in INs. Our findings reveal differences in GABAergic currents between PCs and INs as well as within PC sub-types. We propose that GABAergic inhibition may shape hippocampal output activity by providing cell type-specific fine-tuning of subicular excitatory and inhibitory drives.     

Identification of Meningeal Cell Released Neurite Promoting Activities for Embryonic Hippocampal Neurons

Primary cultures of meningeal cells from embryonic rat cerebra secrete neurite growth-inducing components into serum-free culture medium. This conditioned medium (CM) was analyzed by FPLC and immunochemical and enzymatic treatments and tested for neurite promoting activity (NPA) in a quantitative bioassay using hippocampal neurons from embryonic rat. By immunoprecipitation or specific adsorption we identified laminin (LN)-proteoglycan complexes and fibronectin (FN), respectively, as the major neurite promoting components within meningeal cell CM. The LN-proteoglycan complexes and their NPA were sensitive to chondroitinase (chondroitin ABC lyase, EC 4.2.2.4) and to a smaller extent to heparitinase (heparitin sulfate lyase, EC 4.2.2.8). Minor fractions of the total NPA in CM correlated with free LN and a putative but not yet characterized FN-proteoglycan complex.     

Cell Adhesion Molecules and the Migration of LHRH Neurons during Development

During embryogenesis, LHRH neurons arise in the olfactory epithelium, migrate along the olfactory nerve, and enter the forebrain. We have examined the distribution of several cell adhesion molecules (CAMs) in the developing chick olfactory system and brain to determine whether differential distributions of these adhesion molecules might be important in pathway choices made by migrating LHRH neurons. Single- and double-label immunocytochemical studies indicated that high levels of N-CAM and N-cadherin were expressed throughout the olfactory epithelium and not restricted to the medial half of the olfactory epithelium where most of the LHRH neurons originate. Further, high levels of N-CAM, Ng-CAM, and N-cadherin were uniformly expressed throughout the entire olfactory nerve while migrating LHRH neurons were confined to the medial half of the nerve. However, once LHRH neurons reach the brain, they migrate dorsally and caudally, tangential to the medial surface of the forebrain, along a region enriched in N-CAM and Ng-CAM. After this first stage of migration within the brain, LHRH neurons migrate laterally. At this stage, there is no correlation between the intensity of N-CAM and Ng-CAM immunostaining and the location of LHRH neurons. These results suggest that N-CAM, Ng-CAM, and N-cadherin do not play a guiding role in LHRH neuronal migration through the olfactory epithelium and olfactory nerve but that migrating LHRH neurons may follow a "CAM-trail" of N-CAM and Ng-CAM along the medial surface of the forebrain.     

Regional Distribution and Cell Type-Specific Subcellular Localization of Prothymosin Alpha in Brain

Prothymosin alpha (ProTα) is an acidic nuclear protein implicated in several cellular functions including cell survival. ProTα is found in the central nervous system, but the regional and cell type-specific expression patterns are not known. In this study, our immunohistochemical analysis demonstrated that ProTα is expressed ubiquitously throughout adult brain with difference in the intensity of region-specific protein reactivity. Interestingly, the highest ProTα signals were observed in the brain regions relevant to neurogenesis, such as sub-ventricular zone, granular cell layer of dentate gyrus, as well as granule cell layer of olfactory bulb. Strong immunoreactivity was also found in habenula, ependymal cells lining the dorsal third and fourth ventricle, and in neurons in the Purkinje cell layer of cerebellum. We showed that ProTα was strictly localized in the nuclei of neurons, while it was found in the cytosolic space of astroglial and microglial processes and cell body in the adult brain. To clarify the phenomenon underlying cytosolic localization of ProTα in non-neuronal cells, ZVAD-fmk, a caspase-3 inhibitor, was delivered intracerebroventricularly in the brain. At the follow-up 24 h after ZVAD-fmk injection, we found that nuclear intensity of ProTα was significantly increased in astrocytes, whereas the ProTα expression was not affected in microglia. The present study would contribute toward better understanding of physiological and pathophysiological roles of ProTα in the brain.     

Genetically-Directed,Cell Type-Specific Sparse Labeling for the Analysis of Neuronal Morphology

Background

In mammals, genetically-directed cell labeling technologies have not yet been applied to the morphologic analysis of neurons with very large and complex arbors, an application that requires extremely sparse labeling and that is only rendered practical by limiting the labeled population to one or a few predetermined neuronal subtypes.

Methods and Findings

In the present study we have addressed this application by using CreER technology to non-invasively label very small numbers of neurons so that their morphologies can be fully visualized. Four lines of IRES-CreER knock-in mice were constructed to permit labeling selectively in cholinergic or catecholaminergic neurons [choline acetyltransferase (ChAT)-IRES-CreER or tyrosine hydroxylase (TH)-IRES-CreER], predominantly in projection neurons [neurofilament light chain (NFL)-IRES-CreER], or broadly in neurons and some glia [vesicle-associated membrane protein2 (VAMP2)-IRES-CreER]. When crossed to the Z/AP reporter and exposed to 4-hydroxytamoxifen in the early postnatal period, the number of neurons expressing the human placental alkaline phosphatase reporter can be reproducibly lowered to fewer than 50 per brain. Sparse Cre-mediated recombination in ChAT-IRES-CreER;Z/AP mice shows the full axonal and dendritic arbors of individual forebrain cholinergic neurons, the first time that the complete morphologies of these very large neurons have been revealed in any species.

Conclusions

Sparse genetically-directed, cell type-specific neuronal labeling with IRES-creER lines should prove useful for studying a wide variety of questions in neuronal development and disease.     

Changes of Endocytotic Activities during the Cell Cycle of Dictyostelium Cells

Changes of endocytotic activities during the cell cycle of the cellular slime mould Dictyostelium discoideum Ax-2 were examined using the temperature-shift method for inducing synchronous growth. The activity of fluid-phase pinocytosis (FPP) was altered Ca²⁺-dependently and stimulated by EGTA. On the other hand, pinocytosis was greatly enhanced by addition of Bacteriological-peptone(BP) to the growth medium for Ax-2 cells, irrespective of the extracellular Ca²⁺-concentration. The maximal pinocytotic activity was attained in the presence of EGTA plus BP, the effects of the two substances being additive. The FPP activity was found to be high in cells in and just after the S phase, when the BP-induced fraction of pinocytosis was rather low. Thus the total activity for pinocytosis in the growth medium remained almost constant throughout the cell cycle, indicating that the rate of nutrient uptake through pinocytosis was not a limiting factor for cell cycle regulation. The change of phagocytotic activity during the cell cycle was somewhat similar to that of the FPP activity. Possible mechanisms of such cell-cycle related changes are discussed in relation to cytoskeletal proteins in the cell cortex. Some properties of BP-induced pinocytosis are also described.     

Resolution of Kinase Activities during the HeLa Cell Cycle: Identification of Kinases with Cyclic Activities

Anin situgel kinase assay was used to resolve kinase activities during the HeLa cell cycle. Three kinase activities changed dramatically during the cell cycle, whereas a number of others remained relatively constant. The former included histone kinases with estimated molecular masses of 46, 87, and 39 kDa. The 46-kDa kinase activity was preferentially expressed at least 10-fold greater in mitotic cells. In contrast, the 87- and 39-kDa kinases were most active early in G1 but their activity declined progressively as cells approached mitosis. The 87- but not the 46- or 39-kDa kinases had autophosphorylation activity. The identities of the kinases are unknown but they may play important roles in regulation of the cell cycle.     

Cell Type-Specific Subcellular Localization of Phospho-TBK1 in Response to Cytoplasmic Viral DNA

Cytoplasmic viral RNA and DNA are recognized by RIG-I-like receptors and DNA sensors that include DAI, IFI16, DDX41, and cGAS. The RNA and DNA sensors evoke innate immune responses through the IPS-1 and STING adaptors. IPS-1 and STING activate TBK1 kinase. TBK1 is phosphorylated in its activation loop, leading to IRF3/7 activation and Type I interferon (IFN) production. IPS-1 and STING localize to the mitochondria and endoplasmic reticulum, respectively, whereas it is unclear where phosphorylated TBK1 is localized in response to cytoplasmic viral DNA. Here, we investigated phospho-TBK1 (p-TBK1) subcellular localization using a p-TBK1-specific antibody. Stimulation with vertebrate DNA by transfection increased p-TBK1 levels. Interestingly, stimulation-induced p-TBK1 exhibited mitochondrial localization in HeLa and HepG2 cells and colocalized with mitochondrial IPS-1 and MFN-1. Hepatitis B virus DNA stimulation or herpes simplex virus type-1 infection also induced p-TBK1 mitochondrial localization in HeLa cells, indicating that cytoplasmic viral DNA induces p-TBK1 mitochondrial localization in HeLa cells. In contrast, p-TBK1 did not show mitochondrial localization in RAW264.7, L929, or T-23 cells, and most of p-TBK1 colocalized with STING in response to cytoplasmic DNA in those mammalian cells, indicating cell type-specific localization of p-TBK1 in response to cytoplasmic viral DNA. A previous knockout study showed that mouse IPS-1 was dispensable for Type I IFN production in response to cytoplasmic DNA. However, we found that knockdown of IPS-1 markedly reduced p-TBK1 levels in HeLa cells. Taken together, our data elucidated the cell type-specific subcellular localization of p-TBK1 and a cell type-specific role of IPS-1 in TBK1 activation in response to cytoplasmic viral DNA.     

龙眼果实采后果肉自溶过程中细胞壁组分及其降解酶活性的变化

在(10±1)℃下贮藏的‘福眼’龙眼果实果肉自溶指数和自溶程度随着贮藏时间的延长而增加。果肉细胞壁干重、原果胶、纤维素、半纤维素和细胞壁蛋白含量不断减少。果肉果胶酯酶(PE)活性下降;多聚半乳糖醛酸酶(PG)活性在贮藏6～12d以及纤维素酶活性在贮藏0～12d期间均明显增强,到第12天达到活性高峰,之后下降。但在贮藏0～24d期间,PE、PG和纤维素酶仍然保持较高活性,贮藏24d之后快速下降。β-半乳糖苷酶活性在贮藏0～24d期间略有下降,而在贮藏24d后,活性增强,尤其是贮藏30d后,活性急剧升高。     

Changes in the Autolytic Activities of Maize Coleoptile Cell Walls during Coleoptile Growth

Autolytic activities of coleoptile cell walls were measuredin developing maize seedlings. The major neutral sugar componentsof the cell wall polysaccharides were arabinose, xylose andglucose. The quantities of all these components per coleoptileincreased for 5 d after germination, suggesting that levelsare augmented by biosynthetic processes during coleoptile growth.However, cell wall preparations isolated from the coleoptilesalso revealed increasing rates of autolytic activity directedtoward each of the sugar components. This result suggests thatthe constitutive hydrolytic activities expressed by cell wallsalso increase as a function of coleoptile age. The proportionof glucose in autolysis products relative to that present inthe cell walls specifically increased with coleoptile age, whilethe ratios for arabinose and xylose decreased. Kinetic analysesof autolysis demonstrated that the reactions specific for pentosesat the early growth stage are transient events and that initiallow rates of glucan autolysis increased sharply and persistedlonger. In these experiments the products of glucan autolysiswere largely monomeric while those of the pentose-specific reactionsconsisted of both monomeric and polymeric sugars. Based on theseresults, we concluded that two distinct phases of autolyticactivities are expressed in the mediation of cell wall polysaccharidemetabolism in situ. (Received July 17, 1996; Accepted November 25, 1996)     

Minimal Network Topologies for Signal Processing during Collective Cell Chemotaxis

Cell-cell communication plays an important role in collective cell migration. However, it remains unclear how cells in a group cooperatively process external signals to determine the group’s direction of motion. Although the topology of signaling pathways is vitally important in single-cell chemotaxis, the signaling topology for collective chemotaxis has not been systematically studied. Here, we combine mathematical analysis and simulations to find minimal network topologies for multicellular signal processing in collective chemotaxis. We focus on border cell cluster chemotaxis in the Drosophila egg chamber, in which responses to several experimental perturbations of the signaling network are known. Our minimal signaling network includes only four elements: a chemoattractant, the protein Rac (indicating cell activation), cell protrusion, and a hypothesized global factor responsible for cell-cell interaction. Experimental data on cell protrusion statistics allows us to systematically narrow the number of possible topologies from more than 40,000,000 to only six minimal topologies with six interactions between the four elements. This analysis does not require a specific functional form of the interactions, and only qualitative features are needed; it is thus robust to many modeling choices. Simulations of a stochastic biochemical model of border cell chemotaxis show that the qualitative selection procedure accurately determines which topologies are consistent with the experiment. We fit our model for all six proposed topologies; each produces results that are consistent with all experimentally available data. Finally, we suggest experiments to further discriminate possible pathway topologies.     

Coordination of Rho Family GTPase Activities to Orchestrate Cytoskeleton Responses during Cell Wound Repair

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