Fidelity of the ensemble code for visual motion in primate retina.

Sensory experience typically depends on the ensemble activity of hundreds or thousands of neurons, but little is known about how populations of neurons faithfully encode behaviorally important sensory information. We examined how precisely speed of movement is encoded in the population activity of magnocellular-projecting parasol retinal ganglion cells (RGCs) in macaque monkey retina. Multi-electrode recordings were used to measure the activity of approximately 100 parasol RGCs simultaneously in isolated retinas stimulated with moving bars. To examine how faithfully the retina signals motion, stimulus speed was estimated directly from recorded RGC responses using an optimized algorithm that resembles models of motion sensing in the brain. RGC population activity encoded speed with a precision of approximately 1%. The elementary motion signal was conveyed in approximately 10 ms, comparable to the interspike interval. Temporal structure in spike trains provided more precise speed estimates than time-varying firing rates. Correlated activity between RGCs had little effect on speed estimates. The spatial dispersion of RGC receptive fields along the axis of motion influenced speed estimates more strongly than along the orthogonal direction, as predicted by a simple model based on RGC response time variability and optimal pooling. on and off cells encoded speed with similar and statistically independent variability. Simulation of downstream speed estimation using populations of speed-tuned units showed that peak (winner take all) readout provided more precise speed estimates than centroid (vector average) readout. These findings reveal how faithfully the retinal population code conveys information about stimulus speed and the consequences for motion sensing in the brain.

Quantitation of CD95 and CD95L mRNA expression in chronic and acute HIV-1 infection by competitive RT-PCR.

Human immunodeficiency virus-type 1 (HIV-1) infection is characterized by increased immune cell apoptosis. Apoptosis can be triggered by signals that arise from within the cell, or by signals that are elicited by binding of extracellular "death ligands" to their "death receptors," most of which belong to the tumor necrosis factor (TNF)-receptor family, such as CD95 (Fas/Apo-1). In immune cells the oligomerization of CD95, induced by its ligand CD95L, and the recruitment of different intracytoplasmic molecules that in turn activate FLICE/caspase 8 are crucial. To study the role of CD95/CD95L interactions during HIV-1 infection, we developed an original method based upon quantitative-competitive (QC) RT-PCR that allowed us to quantify the amounts of mRNA coding for the total (tCD95) and membrane (mCD95) forms of CD95. We first studied the expression of different forms of CD95 mRNA in a classical model of chronic HIV infection using two infected cell lines of different origin--lymphocytic (ACH-2) or monocytic (U1). We have shown that infected cells of monocytic origin preferentially produce the "protective" (soluble) form of CD95, and no detectable CD95L mRNA, while lymphoid cells produce more mRNA for the membrane form of CD95 (which triggers apoptosis) along with low but detectable amounts of CD95L mRNA. One can hypothesize that a complex balance exists between pro-apoptotic events, perhaps triggered by the host to limit viral production, and anti-apoptotic events likely triggered by the virus to increase its production and survival. In cells of monocytic origin, which act as a reservoir for the virus, the anti-apoptotic molecules are favored; in cells of lymphocytic origin, molecules with an apoptotic meaning are prevalent.

Differential down-regulation of CD95 or CD95L in chronically HIV-infected cells of monocytic or lymphocytic origin: cellular studies and molecular analysis by quantitative competitive RT-PCR.

We analysed the expression of CD95/CD95L in two widely used models for studying the cellular effects of chronic infection with human immunodeficiency virus type 1 (HIV-1), i.e. ACH-2 cells, derived from the lymphocytic cell line A301, and U1, derived from monocytic U937 cells. A301 and ACH-2 mounted the same amount of plasma membrane CD95, while U1 had a consistent decrease in CD95 expression. Using different antibodies, we failed to detect the plasma membrane form of its ligand, CD95L, but we could see the intracellular presence of that molecule in A301 cells and, to a lesser extent, in ACH-2 cells, but not in U937 or U1 cells. To confirm the cytofluorimetric data and quantify the expression of CD95L at the RNA level, we developed a quantitative competitive RT-PCR assay. The HUT78 cell line had about 50,000 copies mRNA/1000 cells, three times more after induction with a phorbol ester and ionomycin. ACH-2 expressed about 400- (basal) or 10- (induced) fold less CD95L mRNA than the parental cell line A301; U937 and U1 were below the limit of detection. In cells of lymphoid origin (ACH-2) chronic HIV infection inhibits the expression of CD95L, the phenomenon occurring at the transcriptional level. In cells of monocytic origin (U1) the infection decreases the plasma membrane expression of CD95. This suggests that HIV could trigger different anti-apoptotic strategies which likely depend upon the cell line which is infected. In monocytic cells which act as a viral reservoir, the expression of the molecule whose binding triggers apoptosis decreases, while in lymphoid cells, capable of exerting cytotoxicity, the expression of a molecule which induces apoptosis is reduced.

Evaluation of apoptosis of tumor and of apparently normal cells in human renal carcinoma.

Apoptosis of tumor cells and of apparently normal renal cells (ANRC) isolated from the same kidney in 42 untreated patients with renal carcinomas (RC) was evaluated. Thirty five of the investigated tumors were of Grawitz type in different grades of differentiation. The intensity of the apoptotic process was routinely assessed by propidium iodide staining and flow-cytometry analysis. Similar results were obtained in the same cases by using TUNEL assay, by staining with annexin V and by DNA electrophoresis. In 85% of Grawitz carcinomas the proportion of apoptotic tumor cells was quite high, with mean% +/- SD of 57.7+/-27.3, whereas in transitional cell carcinoma of the bladder (TCC), the mean percentage of cells in apoptosis was of 22.3+/-13.9. Unexpectedly, in ANRC displaying normal morphology and normal DNA content (diploidy), the mean% +/- SD of apoptotic cells were found to exceed that of apoptotic tumor cells, 79.2+/-21.6. The percentages of cells expressing Fas receptor and/or Fas ligand varied between large ranges in both tumor and ANRC, thus suggesting that other mechanisms are also involved in the activation of apoptosis. Immunohistochemical studies showed that the intensity of apoptosis correlated well with high p-53 and low bcl-2 expression. The intensity of apoptosis was generally not correlated with the cell proliferation index (S phase fraction), suggesting that in RC apoptosis can be activated in any stage of the cell cycle. Further investigations are necessary to understand the peculiar behaviour of tumor cells as well as of ANRC in renal carcinomas as compared to other types of malignancies.

Optimization of immunochemical methods for intracellular protein detection.

Given the possibility that cell kinetics and p53 status may be important determinants of chemotherapeutical efficacy, the aim of the present study was to determine the optimal methods and conditions for qualitative and quantitative intracellular proteins detection. Bradford assay is the better choice for protein concentration detection because it is more sensitive and more rapid than Sheffield assay despite the fact that it utilizes a higher amount of samples. The direct staining method for flow-cytometrical detection of intracellular proteins is more rapid as compared to the indirect staining one, also providing information about protein expression during cell cycle phases, but it is low sensitive for protein expression estimation and is prohibitive for masked intracellular proteins like PCNA. More than that, it can be performed with both fixed and freshly isolated cells as compared to the indirect staining method, but the last one provides advantages by signal amplification and by its availability of using it for a large number of intracellular proteins detection.