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. Cellwide calcium transients are likely to result from synchronized triggering of such local release events, suggesting that calcium-dependent effector proteins could be selectively activated by localization near sites of local calcium release.

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+. At 200 nM IP3, Ca2+ release was potentiated by an increase in the cytosolic Ca2+ concentration. This potentiation by Ca2+ was nearly absent at 500 nM IP3. Previous studies in smooth muscle cells and neurons showed an inhibition of Ca2+ release above 300 nM Ca2+. In contrast, no such inhibition was observed in RBL cells. When assayed at low cytosolic Ca2+ concentrations, IP3-mediated release was steeply dependent upon luminal Ca2+ concentration. At high luminal Ca2+ concentration, 1 microM IP3 released most of the stored Ca2+ even in the complete absence of cytosolic Ca2+. However, at high cytosolic Ca2+ concentrations (890 nM), IP3-mediated release was no longer steeply dependent upon the luminal Ca2+ concentration. Furthermore, high concentrations of BAPTA inhibited IP3-mediated release in the absence of cytosolic Ca2+. This suggests that a rapid and local luminal Ca2+ feedback is generated by luminal Ca2+ ions binding to cytosolic sites of the same channel or closely associated channels. This "luminal Ca2+ feedback" can be initiated by an increase in the concentration either of IP3, of cytosolic Ca2+, or of luminal Ca2+. It is likely that "luminal Ca2+ feedback" is exploited by cells in both the initiation and termination of Ca2+ spikes.

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 hours post-fertilization (hpf). We show that electroporation is efficient at delivering expression plasmids to large numbers of neurons at multiple developmental steps, including 24, 48, or 96 hours post-fertilization. Expression from electroporated plasmids is maximal within 24 hours, and significant and useful expression is seen within 6 hours. Electroporation can be used to deliver two separate expression plasmids (GFP and mCherry), resulting in co-expression in 97% of cells. Most importantly, electroporation can be used to incorporate siRNA reagents, resulting in 84% knockdown of a target protein (GFP). In conclusion, in vivo electroporation is an effective method for delivering both DNA-based expression plasmids and RNAi-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.

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.

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 1 . In zebrafish, in vivo electroporation has been shown to have excellent spatial and temporal resolution for the delivery of these reagents [2][3][4][5][6][7] . 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 7

INTRODUCTIONIn 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.

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. Genomic resources centered on vertebrate organisms increase daily. As a consequence of optimization of gene transfer protocols by electroporation, the chick embryo will probably become increasingly popular for reverse genetic analysis. The challenge of establishing chick embryonic electroporation might seem insurmountable to those who are unfamiliar with experimental embryological methods. To minimize the cost, time, and effort required to establish a chick electroporation assay method, we describe and illustrate in great detail the procedures involved in building a low-cost electroporation setup and the basic steps of electroporation.

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. Advances in fluorescent Ca2+ imaging have facilitated the resolution of individual positive feedback units. These studies have revealed that there are several modes of channel coupling underlying global Ca2+ signals; single channel openings or Ca2+ "blips," synchronized clusters of channels or Ca2+ "puffs," and cell wide calcium waves. It appears that the channel clusters that produce Ca2+ puffs are synchronized by the highly localized positive feedback that was predicted by the in vitro studies of channel regulation. Localization of InsP3-induced Ca2+ signals has been shown to be important for activation of several cellular processes including uni-directional salt flow and mitochondrial activation.