A new mathematical function to evaluate neuronal morphology using the Sholl analysis.

BACKGROUND: The Sholl analysis is a morphometric method that evaluates the neurite architecture of neurons by drawing a series of concentric circles around the cell soma. Based on the Sholl analysis, one mathematical method that gives a measure of the neurite arborization is the Schoenen ramification index (SRI): the ratio between the maximum number of the intersections of the neurites with the circles and the number of the primary neurites. A different method is the quantification of the number of bifurcations of the neurites (BN). NEW METHOD: In this study we proposed a new mathematical function to quantify neurite morphology that we named the branching index (BI). The BI compares the difference in the number of intersections made in pairs of circles relative to the distance from the neuronal soma. To facilitate the morphometric analysis, we developed informatics software named CellTarget that obtains the quantitative variables of the Sholl analysis and neurite branching. RESULTS: Using that bioinformatics application we compared the BI, the SRI and BN values in neuronal models and in neuronal hippocampal cultures treated or untreated with the androgen dihydrotestosterone, which is known to induce neurite branching. COMPARISON WITH EXISTING METHODS: Although the SRI and the BN provided quantitative information of the degree of neurite morphology, it produced similar values in neurons that ramify very differently. By contrast, these differences were discriminated using the BI. CONCLUSIONS: The BI is a useful parameter to discriminate among different neuronal morphologies.

The cholecystokinin system in the rat retina: receptor expression and in vivo activation of tyrosine phosphorylation pathways.

High levels of endogenous cholecystokinin (CCK) are present in the rat retina and restricted to amacrine cells. Two types of CCK receptors exist but their expression and intracellular transduction pathways coupled in the rat retina are unknown. The aims of this study were to investigate CCK receptors expression in rat retina and to study downstream tyrosine phosphorylation pathways. For this purpose, total mRNA isolated from rat retina was subjected to RT-PCR analysis. Isolated rat retinas were incubated in presence of CCK. Soluble proteins in retinal homogenates were immunoprecipitated with anti-phoshpotyrosine or anti-p130(Cas) specific monoclonal antibodies and subjected to SDS-PAGE, followed by Western blotting analysis. Both types of CCK receptor mRNAs, A and B, are present in the rat retina. Incubation of retina with CCK induced a rapid increase in several phosphotyrosine-containing bands with molecular masses greater than 30 kDa. Western Blotting and immunoprecipitation with a specific monoclonal antibody identified one of the phosphotyrosine bands as the adapter protein p130(Cas). Tyrosine phosphorylation of p130(Cas) induced by CCK in rat retina was time and concentration dependent: CCK induced tyrosine phosphorylation of p130(Cas) occurred rapidly with the maximum effect observed at 2.5 min incubation with 1 microM CCK. Our data clearly identified CCK-A and CCK-B receptor mRNAs in the rat retina and demonstrated that they are functional, stimulating tyrosine phosphorylation pathways. Our results provide novel biochemical information to further understand the physiological role of CCK A and B receptors in rat retina.

Cloning, expression, and regulation by androgens of a putative member of the oxytocinase family of proteins in the rat prostate.

BACKGROUND: Proteases are relevant in the physiology of the prostate, and its expression is regulated by androgens. METHODS: Isolation of a novel cDNA from the rat prostate was done by reverse transcriptase-polymerase chain reaction and rapid amplification of cDNA ends. By Northern blot, we analyzed the RNA expression in different tissues and in the prostate after orchidectomy and androgen treatment. By using in situ hybridization, we studied the cellular localization of the RNA. RESULTS: The cDNA codes a putative novel form of the vp-165 aminopeptidase family of proteins that we named short-vp. The short-vp probe labels one mRNA of 1.3 kb in the prostate, brain, testis, heart, and kidney. In the prostate, the levels of short-vp mRNA decrease after orchidectomy and increase with testosterone treatment. The short-vp mRNA is expressed by the prostatic epithelial cells. CONCLUSION: We isolated one putative member of the oxytocinase family of proteins that is expressed in various tissues and by the epithelial cells of the prostate. The expression of short-vp mRNA in the prostate depends on androgen levels.

Cloning of the cDNA and mRNA expression of CLRP, a complex leucine repeat protein of the Golgi apparatus expressed by specific neurons of the rat brain.

We report the molecular cloning of one novel cDNA isolated from the rat brain. We have named the putative protein CLRP, for complex leucine-repeat protein. The predicted CLRP amino acid sequence shares homology in the amino acid composition with the Galactose, N-Acetylglucosamine, and Sialic acid transporters, and shows 91% identity with the sequence of one human chromosome 5 BAC clone. Expression of the CLRP cDNA tagged with GFP in COS-7 cells was found in cell organelles that resemble the Golgi apparatus of the cytoplasm. In Northern blot, the CLRP probe labels a single band of 2.4 kb in the brain, kidney, lung, testis, and prostate. In the brain, CLRP mRNA is expressed by limited sets of neurons, such as the pyramidal cells of the cortex, the Purkinje cells of the cerebellum, and the motoneurons of the brainstem. In the brain, the CLRP mRNA is expressed at embryonic day 15; levels of expression are maintained until postnatal day 10 and decrease in adults. The results suggest that CLRP codes a novel member of the nucleotide-sugar family of proteins of the Golgi apparatus.