Teaching Basic Calculations in an Introductory Biology Lab.

Quantitative reasoning is one of the core competencies identified as a priority for transforming the undergraduate biology curriculum. However, first-year biology majors often lack confidence in their quantitative skills. We revised an introductory biology lab to emphasize the teaching of basic laboratory calculations, utilizing multiple teaching tools, including online prelab quizzes, minilab lectures, calculation worksheets, and online video tutorials.

A detailed description of an economical setup for electroporation of chick embryos in ovo.

One of the challenges of the postgenomic era is characterizing the function and regulation of specific genes. For various reasons, the early chick embryo can easily be adopted as an in vivo assay of gene function and regulation. The embryos are robust, accessible, easily manipulated, and maintained in the laboratory.

Targeting olfactory bulb neurons using combined in vivo electroporation and Gal4-based enhancer trap zebrafish lines.

In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific regions of developing embryos, including those of C. elegans, chick, Xenopus, zebrafish, and mouse. In zebrafish, in vivo electroporation has been shown to have excellent spatial and temporal resolution for the delivery of these reagents.

Real-time measurement of face recognition in rapid serial visual presentation.

Event-related potentials (ERPs) have been used extensively to study the processes involved in recognition memory. In particular, the early familiarity component of recognition has been linked to the FN400 (mid-frontal negative deflection between 300 and 500 ms), whereas the recollection component has been linked to a later positive deflection over the parietal cortex (500-800 ms). In this study, we measured the ERPs elicited by faces with varying degrees of familiarity.

Targeting the zebrafish optic tectum using in vivo electroporation.

In vivo electroporation is a method for delivery of plasmids and other oligonucleotide reagents that offers precise temporal control. In zebrafish, in vivo electroporation is particularly well-suited to delivering green fluorescent protein (GFP) expression vectors to the developing central nervous system. This protocol describes a modification of in vivo electroporation that can be used to specifically target the developing optic tectum of zebrafish embryos beginning at 24 h post-fertilization (hpf).

The temporal resolution of in vivo electroporation in zebrafish: a method for time-resolved loss of function.

One caveat to current loss-of-function approaches in zebrafish is that they typically disrupt gene function from the beginning of development. This can be problematic when attempting to study later developmental events. In vivo electroporation is a method that has been shown to be effective at incorporating reagents into the developing nervous system at multiple later developmental stages.

Expert image analysts show enhanced visual processing in change detection.

Expertise facilitates change detection performance, but the neural underpinnings of these benefits are unknown. Expert image analysts showed larger change-related ERP effects between about 100-200 msec after stimulus onset than did novices, which correlated with both accuracy and years of analysis experience. These results demonstrate that years of visual experience can induce fundamental changes in early visual processing which are related to change detection abilities.

Integration of calcium signals by calmodulin in rat sensory neurons.

We have used the fluorescently labelled calmodulin TA-CaM to follow calmodulin activation during depolarization of adult rat sensory neurons. Calcium concentration was measured simultaneously using the low affinity indicator Oregon Green BAPTA 5N. TA-CaM fluorescence increased during a 200-ms depolarization but then continued to increase during the subsequent 500 ms, even though total cell calcium was falling at this time.

Regulatory and spatial aspects of inositol trisphosphate-mediated calcium signals.

Hormones that act to release Ca2+ from intracellular stores initiate a signaling cascade that culminates in the production of inositol 1,4,5-trisphosphate (InsP3). The Ca2+ response mediated by InsP3 is not a sustained increase in the cytosolic Ca2+ concentration, but rather a series of periodic spikes that manifest as waves in larger cells. In vitro studies have determined that the key positive feedback parameter driving spikes and waves is a highly localized direct Ca(2+)-activation of InsP3-gated Ca2+ channels.

Characterization of a dextran-based bifunctional calcium indicator immobilized in cells by the enzymatic addition of isoprenoid lipids.

Cellular processes can be controlled by cell-wide increases in the cytosolic Ca2+ concentration or, alternatively, by localized Ca2+ signals in micro- and nano-domains. The experimental characterization of such localized Ca2+ signals would be facilitated using an immobilized Ca2+ indicator, which could prevent the accelerated spatial spreading of Ca2+ ions that is mediated by binding to diffusible indicators. Here we characterize a dextran-based Ca2+ indicator (CAAX-green) that becomes immobilized in the cytosol by an enzyme-mediated addition of a geranylgeranyl lipid group.

Elementary calcium-release units induced by inositol trisphosphate.

The extent to which inositol 1,4,5-trisphosphate (InsP3)-induced calcium signals are localized is a critical parameter for understanding the mechanism of effector activation. The spatial characteristics of InsP3-mediated calcium signals were determined by targeting a dextran-based calcium indicator to intracellular membranes through the in situ addition of a geranylgeranyl lipid group. Elementary calcium-release events observed with this indicator typically lasted less than 33 milliseconds, had diameters less than 2 micrometers, and were uncoupled from each other by the calcium buffer EGTA.

Luminal calcium regulates the inositol trisphosphate receptor of rat basophilic leukemia cells at a cytosolic site.

Hormones, growth factors, and other stimuli can generate Ca2+ spikes and waves by activation of the phosphoinositide (PI) pathway. The sources of these Ca2+ signals are inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ stores. Here we use a rapid perfusion apparatus to measure the release of 45Ca2+ from permeabilized rat basophilic leukemia (RBL) cells to investigate the regulation of IP3-mediated Ca2+ release by cytosolic and luminal Ca2+.

Effects of recombinant human granulocyte-macrophage colony-stimulating factor on intracellular pH in mature granulocytes.

We studied the effects of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSFrh) on the internal pH of granulocytes using the fluorescent probe BCECF. GM-CSFrh did not directly alter the resting pH of granulocytes isolated from the peripheral blood; however, when the cells were preincubated for 90 minutes with the growth factor and then activated with the chemotactic peptide N-formyl met leu phe (fMLP), they exhibited both an acceleration in the initial rate of acidification and a marked delay in realkalinization. The kinetic changes both in initial acidification and in subsequent realkalinization induced by GM-CSFrh priming were not prevented by protein synthesis inhibitors and were observed in granulocytes harvested from patients with both sex-linked and autosomal recessive chronic granulomatous disease (CGD).