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1.
PLoS Comput Biol ; 13(6): e1005578, 2017 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-28617798

RESUMEN

Neural coding through inhibitory projection pathways remains poorly understood. We analyze the transmission properties of the Purkinje cell (PC) to cerebellar nucleus (CN) pathway in a modeling study using a data set recorded in awake mice containing respiratory rate modulation. We find that inhibitory transmission from tonically active PCs can transmit a behavioral rate code with high fidelity. We parameterized the required population code in PC activity and determined that 20% of PC inputs to a full compartmental CN neuron model need to be rate-comodulated for transmission of a rate code. Rate covariance in PC inputs also accounts for the high coefficient of variation in CN spike trains, while the balance between excitation and inhibition determines spike rate and local spike train variability. Overall, our modeling study can fully account for observed spike train properties of cerebellar output in awake mice, and strongly supports rate coding in the cerebellum.


Asunto(s)
Potenciales de Acción/fisiología , Núcleos Cerebelosos/fisiología , Modelos Neurológicos , Red Nerviosa/fisiología , Inhibición Neural/fisiología , Células de Purkinje/fisiología , Animales , Simulación por Computador , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Vías Nerviosas/fisiología , Vigilia/fisiología
2.
J Neurophysiol ; 114(2): 1346-52, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-26108956

RESUMEN

Chondroitin sulfate proteoglycans (CSPGs) are widely studied in vertebrate systems and are known to play a key role in development, plasticity, and regulation of cortical circuitry. The mechanistic details of this role are still elusive, but increasingly central to the investigation is the homeostatic balance between network excitation and inhibition. Studying a simpler neuronal circuit may prove advantageous for discovering the mechanistic details of the cellular effects of CSPGs. In this study we used a well-established model of homeostatic change after injury in the crab Cancer borealis to show first evidence that CSPGs are necessary for network activity homeostasis. We degraded CSPGs in the pyloric circuit of the stomatogastric ganglion with the enzyme chondroitinase ABC (chABC) and found that removal of CSPGs does not influence the ongoing rhythm of the pyloric circuit but does limit its capacity for recovery after a networkwide perturbation. Without CSPGs, the postperturbation rhythm is slower than in controls and rhythm recovery is delayed. In addition to providing a new model system for the study of CSPGs, this study suggests a wider role for CSPGs, and perhaps the extracellular matrix in general, beyond simply plastic reorganization (as observed in mammals) and into a foundational regulatory role of neural circuitry.


Asunto(s)
Proteínas de Artrópodos/metabolismo , Braquiuros/fisiología , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Factor de Crecimiento Epidérmico/deficiencia , Espacio Extracelular/metabolismo , Factor I del Crecimiento Similar a la Insulina/deficiencia , Animales , Far-Western Blotting , Condroitina ABC Liasa/metabolismo , Factor de Crecimiento Epidérmico/fisiología , Ganglios de Invertebrados/metabolismo , Insulina/deficiencia , Factor I del Crecimiento Similar a la Insulina/fisiología , Errores Innatos del Metabolismo , Periodicidad , Técnicas de Cultivo de Tejidos , Síndrome de Werner
3.
Am J Physiol Renal Physiol ; 300(2): F475-87, 2011 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-21048021

RESUMEN

Vitronectin (VN), secreted into the bloodstream by liver hepatocytes, is known to anchor epithelial cells to basement membranes through interactions with cell surface integrin receptors. We report here that VN is also synthesized by urothelial cells of urothelium in vivo and in vitro. In situ hybridization, dideoxy sequencing, immunohistochemistry, and ELISA of urothelial cell mRNA, cDNA, tissue, and protein extracts demonstrated that the VN gene is active in vivo and in vitro. The expression of VN by urothelium is hypothesized to constitute one of several pathways that anchor basal cells to an underlying substratum and explains why urothelial cells adhere to glass and propagate under serum-free conditions. Therefore, two sources of VN in the human urinary bladder are recognized: 1) localized synthesis by urothelial cells and 2) extravasation of liver VN through fenestrated capillaries. When human plasma was fractionated by denaturing heparin affinity chromatography, VN was isolated in a biologically active form that supported rapid spreading of urothelial cells in vitro under serum-free conditions. This activity was inhibited by the matricellular protein SPARC via direct binding of VN to SPARC through a Ca(+2)-dependent mechanism. A novel form of VN, isolated from the same heparin affinity chromatography column and designated as the VN(c) chromatomer, also supported cell spreading but failed to interact with SPARC. Therefore, the steady-state balance among urothelial cells, their extracellular milieu, and matricellular proteins constitutes a principal mechanism by which urothelia are anchored to an underlying substrata in the face of constant bladder cycling.


Asunto(s)
Sistema Urinario/metabolismo , Urotelio/metabolismo , Vitronectina/biosíntesis , Adolescente , Secuencia de Aminoácidos , Secuencia de Bases , Calcio/metabolismo , Células Cultivadas , Niño , Preescolar , Matriz Extracelular/metabolismo , Humanos , Lactante , Recién Nacido , Datos de Secuencia Molecular , Sistema Urinario/citología , Vitronectina/genética
4.
Physiology (Bethesda) ; 25(5): 311-8, 2010 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-20940436

RESUMEN

Neural circuits rely on slight physiological differences between the component cells for proper function. When any circuit is analyzed, it is important to characterize the features that distinguish one cell type from another. This review describes the methods used to identify the neurons of the crustacean stomatogastric ganglion.


Asunto(s)
Crustáceos/fisiología , Ganglios de Invertebrados/fisiología , Neuronas/fisiología , Estómago/inervación , Estómago/fisiología , Animales , Ganglios de Invertebrados/citología
5.
PLoS Comput Biol ; 6(7): e1000838, 2010 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-20628472

RESUMEN

Recent experimental evidence suggests that coordinated expression of ion channels plays a role in constraining neuronal electrical activity. In particular, each neuronal cell type of the crustacean stomatogastric ganglion exhibits a unique set of positive linear correlations between ionic membrane conductances. These data suggest a causal relationship between expressed conductance correlations and features of cellular identity, namely electrical activity type. To test this idea, we used an existing database of conductance-based model neurons. We partitioned this database based on various measures of intrinsic activity, to approximate distinctions between biological cell types. We then tested individual conductance pairs for linear dependence to identify correlations. Contrary to experimental evidence, in which all conductance correlations are positive, 32% of correlations seen in this database were negative relationships. In addition, 80% of correlations seen here involved at least one calcium conductance, which have been difficult to measure experimentally. Similar to experimental results, each activity type investigated had a unique combination of correlated conductances. Finally, we found that populations of models that conform to a specific conductance correlation have a higher likelihood of exhibiting a particular feature of electrical activity. We conclude that regulating conductance ratios can support proper electrical activity of a wide range of cell types, particularly when the identity of the cell is well-defined by one or two features of its activity. Furthermore, we predict that previously unseen negative correlations and correlations involving calcium conductances are biologically plausible.


Asunto(s)
Modelos Neurológicos , Neuronas Motoras/fisiología , Conducción Nerviosa/fisiología , Potenciales de Acción , Animales , Canales de Calcio/fisiología , Distribución de Chi-Cuadrado , Crustáceos , Bases de Datos Factuales , Tracto Gastrointestinal/inervación
6.
Tissue Eng ; 13(9): 2219-25, 2007 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-17638517

RESUMEN

Synthetic urothelium is an important goal for the tissue-engineering field that would have great utility for treating diseases and congenital defects affecting the urinary tract. A key step in the development of synthetic tissue is optimizing the conditions for coating biomaterials with cells of interest. Initial cell attachment is an important consideration when designing tissue-engineering scaffolds. The scaffold environment must also be conducive to cell proliferation and differentiation. The most popular materials for tissue-engineering scaffold often have suboptimal properties when analyzed for cell attachment and growth. It would then be of interest to know, for urinary tract tissue-engineering applications, which extracellular matrix protein coatings can facilitate urothelial cell attachment and encourage growth. Cells grown on 96-well cycloolefin plates coated with type IV or type I collagen exhibited improved initial attachment over plates coated with fibronectin or laminin. After 20 h, deoxyribonucleic acid synthesis was found to increase in cultures grown on type IV collagen, fibronectin, and laminin. Total metabolic activity of urothelial cell cultures was also monitored, and no difference was seen between any protein-coating conditions. The development of such reliable assays will be beneficial in monitoring the fate of scaffolds seeded with human urothelial cells.


Asunto(s)
Materiales Biocompatibles Revestidos , Células Epiteliales/fisiología , Proteínas de la Matriz Extracelular , Urotelio/citología , Adhesión Celular/fisiología , Células Cultivadas , Colágeno Tipo I/metabolismo , Colágeno Tipo IV/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Humanos , Laminina/metabolismo , Unión Proteica , Ingeniería de Tejidos , Urotelio/metabolismo
7.
J Cell Physiol ; 206(1): 211-20, 2006 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-16121393

RESUMEN

The anti-spreading activity of secreted protein acidic and rich in cysteine (SPARC) has been assigned to the C-terminal third domain, a region rich in alpha-helices. This "extracellular calcium-binding" (EC) domain contains two EF-hands that each coordinates one Ca2+ ion, forming a helix-loop-helix structure that not only drives the conformation of the protein but is also necessary for biological activity. Recombinant (r) EC, expressed in E. coli, was fused at the C-terminus to a His hexamer and isolated under denaturing conditions by nickel-chelate affinity chromatography. rEC-His was renatured by procedures that simultaneously (i) removed denaturing conditions, (ii) catalyzed disulfide bond isomerization, and (iii) initiated Ca2+-dependent refolding. Intrinsic tryptophan fluorescence and circular dichroism spectroscopies demonstrated that rEC-His exhibited a Ca2+-dependent conformation that was consistent with the known crystal structure. Spreading assays confirmed that rEC-His was biologically active through its ability to inhibit the spreading of freshly plated human urothelial cells propagated from transitional epithelium. rEC-His and rSPARC-His exhibited highly similar anti-spreading activities when measured as a function of concentration or time. In contrast to the wild-type and EC recombinant proteins, rSPARC(E268F)-His, a point substitution mutant at the Z position of EF-hand 2, failed to exhibit both Ca2+-dependent changes in alpha-helical secondary structure and anti-spreading activity. The collective data provide evidence that the motif of SPARC responsible for anti-spreading activity was dependent on the coordination of Ca2+ by a Glu residue at the Z position of EF-hand 2 and provide insights into how adhesive forces are balanced within the extracellular matrix of urothelial cells. .


Asunto(s)
Movimiento Celular/fisiología , Osteonectina/metabolismo , Urotelio/citología , Adolescente , Secuencia de Aminoácidos , Calcio/metabolismo , Escherichia coli , Femenino , Humanos , Lactante , Datos de Secuencia Molecular , Osteonectina/química , Osteonectina/genética , Conformación Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo
8.
J Cell Physiol ; 202(2): 453-63, 2005 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-15389586

RESUMEN

The AON epitope of secreted protein acidic and rich in cysteine (SPARC) is a conserved motif expressed by human SPARC in a variety of human cell types. Through the use of a monoclonal antibody that recognizes this epitope, transitional epithelium was found to restrict expression of SPARC to the suprabasal and intermediate layer. Such intracellular expression was defined by immunoreactive signals that localized to the apical plasma membranes of suprabasal and intermediate cells. Polarization of SPARC to apical plasma membranes of suprabasal cells was retained in vitro by a subpopulation of cells that exhibited characteristics of suprabasal cells--cell-cycle quiescence, large cell volumes, and multiple nuclei. In contrast, the basal layer of transitional epithelium in vivo and cycling cells in vitro did not exhibit this apical staining pattern, but instead sequestered the SPARC polypeptide within urothelial cytoplasm and/or nuclei, as revealed by immunohistochemical analysis. Elution of soluble proteins and DNA from urothelial cells revealed the presence of SPARC within the nuclear matrix--and that SPARC colocalized with the nuclear matrix Ki-67 antigen. rSPARC activity was demonstrated and quantified with a rounding assay whereby the spreading of freshly plated cells was inhibited by recombinant SPARC in a concentration- and time-dependent manner. Inhibition of spreading was observed in urothelial cells derived from endoderm (bladder) and mesoderm (ureter) germ layers. Statistically significant differences were seen between urothelial cells from these two layers. Mesodermal cells recovered more slowly from the inhibitory effects of rSPARC, such that at hour 6 endodermal cells underwent significantly more spreading, as shown by a rounding index (RI). These experiments provide new insights about the matricellular trafficking of SPARC and suggest that intra- and extra-cellular localization patterns influence the development, homeostasis, and differentiation of transitional epithelium.


Asunto(s)
Osteonectina/fisiología , Uréter/citología , Uréter/fisiología , Vejiga Urinaria/citología , Vejiga Urinaria/fisiología , Células Cultivadas , Endodermo/citología , Endodermo/efectos de los fármacos , Endodermo/fisiología , Humanos , Inmunohistoquímica , Mesodermo/citología , Mesodermo/efectos de los fármacos , Mesodermo/fisiología , Matriz Nuclear/metabolismo , Osteonectina/química , Osteonectina/farmacología , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Uréter/efectos de los fármacos , Uréter/embriología , Vejiga Urinaria/efectos de los fármacos , Vejiga Urinaria/embriología , Urotelio/citología , Urotelio/efectos de los fármacos , Urotelio/embriología , Urotelio/fisiología
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