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. In addition, we implemented a repetitive assessment approach whereby three types of basic calculations-unit conversions, calculating molar concentrations, and calculating dilutions-were assessed on all quizzes and exams throughout the semester. The results showed that learning improved for each of the three quantitative problem types assessed and that these learning gains were statistically significant, both from first assessment to midterm and, notably, from midterm to final. Additionally, the most challenging problem type for students, calculating molar concentrations, showed the greatest normalized learning gains in the second half of the semester. The latter result suggests that persistent assessment resulted in continued learning even after formal, in-class teaching of these approaches had ended. This approach can easily be applied to other courses in the curriculum and, given the learning gains achieved, could provide a powerful means to target other quantitative skills.

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. The temporal resolution of this method is important because it allows for incorporation of these reagents at specific stages in development. Furthermore, because expression from electroporated vectors occurs within 6 hours, this method is more timely than transgenic approaches. While the spatial resolution can be extremely precise when targeting a single cell, it is often preferable to incorporate reagents into a specific cell population within a tissue or structure. When targeting multiple cells, in vivo electroporation is efficient for delivery to a specific region of the embryo; however, particularly within the developing nervous system, it is difficult to target specific cell types solely through spatially discrete electroporation. Alternatively, enhancer trap transgenic lines offer excellent cell type-specific expression of transgenes. Here we describe an approach that combines transgenic Gal4-based enhancer trap lines with spatially discrete in vivo electroporation to specifically target developing neurons of the zebrafish olfactory bulb. The Et(zic4:Gal4TA4,UAS:mCherry)(hzm5) (formerly GA80_9) enhancer trap line previously described, displays targeted transgenic expression of mCherry mediated by a zebrafish optimized Gal4 (KalTA4) transcriptional activator in multiple regions of the developing brain including hindbrain, cerebellum, forebrain, and the olfactory bulb. To target GFP expression specifically to the olfactory bulb, a plasmid with the coding sequence of GFP under control of multiple Gal4 binding sites (UAS) was electroporated into the anterior end of the forebrain at 24-28 hours post-fertilization (hpf). Although this method incorporates plasmid DNA into multiple regions of the forebrain, GFP expression is only induced in cells transgenically expressing the KalTA4 transcription factor. Thus, by using the GA080_9 transgenic line, this approach led to GFP expression exclusively in the developing olfactory bulb. GFP expressing cells targeted through this approach showed typical axonal projections, as previously described for mitral cells of the olfactory bulb. This method could also be used for targeted delivery of other reagents including short-hairpin RNA interference expression plasmids, which would provide a method for spatially and temporally discrete loss-of-function analysis.

Targeting the zebrafish optic tectum using in vivo electroporation.

INTRODUCTION: 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 electroporation electrodes required for this approach can be constructed easily from relatively inexpensive materials. Microinjection of plasmid DNA to the midbrain ventricle followed by precise positioning of the electroporation electrodes allows for the targeting of developing neurons in only one hemisphere of the optic tectum. Using this protocol, the optic tectum can be effectively targeted in a high percentage (79%) of expressing embryos. This method can also be used to simultaneously deliver expression vectors and loss-of-function reagents, which can provide precise temporal control of the knockdown of gene function.

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. The temporal and spatial characteristics of in vivo electroporation that have been previously demonstrated suggest that this could be a powerful approach for time-resolved loss-of-function analysis. Here, in an attempt to demonstrate the efficacy of this approach for analysis of a specific developmental timeframe--that of initial development of the zebrafish visual system-we have done a systematic characterization of the efficiency of in vivo electroporation in zebrafish across multiple developmental stages, from 24 to 96 h postfertilization. We show that electroporation is efficient at delivering expression plasmids to large numbers of neurons at multiple developmental steps, including 24, 48, or 96 h postfertilization. Expression from electroporated plasmids is maximal within 24 h, and significant and useful expression is seen within 6 h. Electroporation can be used to deliver two separate expression plasmids (green fluorescent protein and mCherry), resulting in coexpression in 97% of cells. Most importantly, electroporation can be used to incorporate siRNA reagents, resulting in 84% knockdown of a target protein (green fluorescent protein). In conclusion, in vivo electroporation is an effective method for delivering both DNA-based expression plasmids and RNA interference-based loss-of-function reagents, and exhibits the appropriate characteristics to be useful as a time-resolved genetic approach to investigate the molecular mechanisms of visual system development.

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. In the next few seconds TA-CaM fluorescence fell, but to a new elevated level that was then maintained for several tens of seconds. During a train of depolarizations that evoked a series of largely independent calcium changes TA-CaM fluorescence was in contrast raised for the duration of the train and for many tens of seconds afterwards. The presence of a peptide corresponding to the calmodulin binding domain of myosin light chain kinase significantly increased the depolarization-induced TA-CaM fluorescence increase and slowed the subsequent fall of fluorescence. We interpret the slow recovery component of the TA-CaM signal as reflecting the slow dissociation of calcium--calmodulin--calmodulin binding protein complexes. Our results show that after brief electrical activity calmodulin's interaction with calmodulin binding proteins persists for approximately one minute.