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Multicellular glial calcium waves may locally regulate neural activity or brain energetics. Here, we report a diffusion-driven astrocytic signal in the normal, intact brain that spans many astrocytic processes in a confined volume without fully encompassing any one cell. By using 2-photon microscopy in rodent cerebellar cortex labeled with fluorescent indicator dyes or the calcium-sensor protein G-CaMP2, we discovered spontaneous calcium waves that filled approximately ellipsoidal domains of Bergmann glia processes. Waves spread in 3 dimensions at a speed of 4-11[micro]m/s to a diameter of [approximately equal to] 50 [micro]m, slowed during expansion, and were reversibly blocked by P2 receptor antagonists. Consistent with the hypothesis that ATP acts as a diffusible trigger of calcium release waves, local ejection of ATP triggered P2 receptor-mediated waves that were refractory to repeated activation. Transglial waves represent a means for purinergic signals to act with local specificity to modulate activity or energetics in local neural circuits. astrocytes | Bergmann glia | in vivo | 2-photon microscopy | G-CaMP2

Accumulating evidence indicates that plasma membrane (PM) microdomains and the subjacent 'junctional' sarcoplasmic/endoplasmic reticulum (jS/ER) constitute specialized [Ca.sup.2+] signaling complexes in many cell types. We examined the possibility that some [Ca.sup.2+] signals arising in the junctional space between the PM and jS/ER may represent cross-talk between the PM and jS/ER. The [Ca.sup.2+] sensor protein, GCaMP2, was targeted to different PM domains by constructing genes for fusion proteins with either the [alpha]1 or [alpha]2 isoform of the [Na.sup.+]pump catalytic ([alpha]) subunit. These fusion proteins were expressed in primary cultured mouse brain astrocytes and arterial smooth muscle cells. Immunocytochemistry demonstrated that [alpha]2(f)GCaMP2, like native [Na.sup.+] pumps with [alpha]2-subunits, sorted to PM domains that colocalized with subjacent S/ER, [alpha]1(f)GCaMP2, like [Na.sup.+] pumps with [alpha]1-subunits, was more uniformly distributed. The GCaMP2 moieties in both constructs were tethered just beneath the PM. Both constructs detected global [Ca.sup.2+] signals evoked by serotonin (in arterial smooth muscle cells) and ATP, and by store-operated [Ca.sup.2+] channel-mediated [Ca.sup.2+] entry after S/ER unloading with cyclopiazonic acid (in [Ca.sup.2+]-free medium). When cytosolic [Ca.sup.2+] diffusion was markedly restricted with EGTA, however, only [alpha]2(f)GCaMP2 detected the local, storeoperated [Ca.sup.2+] channel-mediated [Ca.sup.2+] entry signal. Thus, [alpha]1 [Na.sup.+] pumps are apparently excluded from the PM microdomains occupied by [alpha]2 [Na.sup.+] pumps. The jS/ER and adjacent PM may communicate by [Ca.sup.2+] signals that are confined to the tiny junctional space between the two membranes. Similar methods may be useful for studying localized [Ca.sup.2+] signals in other subPM microdomains and signals associated with other organelles. arterial smooth muscle | astrocytes | [Na.sup.+] pump | PLasmERosome | store-operated [Ca.sup.2-] entry

Genetically encoded sensor proteins provide unique opportunities to advance the understanding of complex cellular interactions in physiologically relevant contexts; however, previously described sensors have proved to be of limited use to report cell signaling in vivo in mammals. Here, we describe an improved [Ca.sup.2+] sensor, GCaMP2, its inducible expression in the mouse heart, and its use to examine signaling in heart cells in vivo. The high brightness and stability of GCaMP2 enable the measurement of myocyte [Ca.sup.2+] transients in all regions of the beating mouse heart and prolonged pacing and mapping studies in isolated, perfused hearts. Transgene expression is efficiently temporally regulated in cardiomyocyte GCaMP2 mice, allowing recording of in vivo signals 4 weeks after transgene induction. High-resolution imaging of [Ca.sup.2+] waves in GCaMP2-expressing embryos revealed key aspects of electrical conduction in the preseptated heart. At embryonic day (e.d.) 10.5, atrial and ventricular conduction occur rapidly, consistent with the early formation of specialized conduction pathways. However, conduction is markedly slowed through the atrioventricular canal in the e.d. 10.5 heart, forming the basis for an effective atrioventricular delay before development of the AV node, as rapid ventricular activation occurs after activation of the distal AV canal tissue. Consistent with the elimination of the inner AV canal muscle layer at e.d. 13.5, atrioventricular conduction through the canal was abolished at this stage. These studies demonstrate that GCaMP2 will have broad utility in the dissection of numerous complex cellular interactions in mammals, in vivo. atrioventricular node | [Ca.sup.2+] imaging | genetic sensor | heart development | fluorescent [Ca.sup.2+] sensor

G-CaMP is a [Ca.sup.2+] probe based on a single green fluorescent protein (GFP). G-CaMP shows a large fluorescence increase upon [Ca.sup.2+] binding, but its fluorescence is dim and pH sensitive, similar to other single GFP-based probes. Here we report an improved G-CaMP, named G-CaMP1.6, which enables easier detection of intracellular [Ca.sup.2+] signals. G-CaMP1.6 was ~40 times more fluorescent than G-CaMP, mainly due to an increase in quantum yield. Furthermore, compared with G-CaMP, G-CaMP1.6 had not only a lower pH sensitivity but also a higher selectivity for divalent cations having an ionic radius similar to [Ca.sup.2+]. [Ca.sup.2+] sensitivity of G-CaMP1.6 ([K.sub.d] = 146 nM, Hill coefficient = 3.8, [F.sub.max]/[F.sub.min] = 4.9) was slightly shifted toward higher affinity compared with that of G-CaMP. When expressed in mammalian cells, G-CAMP1.6 showed large fluorescence changes with drug applications. Notably, local [Ca.sup.2+] changes in such tiny structures as dendritic spines of neurons were successfully observed with G-CaMP1.6, this being the first observation using a GFP-based probe. Additional mutations in [Ca.sup.2+]-binding sites of G-CaMP1.6 shifted the affinity for [Ca.sup.2+] and reduced the [Ca.sup.2+]-buffering effect. G-CaMP1.6-CaM(E140K), which has a mutation in the [Ca.sup.2+] binding site, is an improved probe with its increased brightness and reduced [Ca.sup.2+]-buffering capacity.

The silkworm Bombyx mori is one of the most well-studied insects in terms of both genetics and physiology and is recognized as the model lepidopteran insect. To develop an efficient system for analyzing gene function in the silkworm, we investigated the feasibility of using the GAL4/UAS system in conjunction with piggyBac vector-mediated germ-line transformation for targeted gene expression. To drive the GAL4 gene, we used two endogenous promoters that originated from the B. mori actin A3 (BmA3) and fibroin lightchain (FiL) genes and the artificial promoter 3xP3. GFP was used as the reporter. In initial tests of the function of the GAL4/UAS system, we generated transgenic animals that carried the UAS-GFP construct plus either BmA3-GAL4 or 3xP3-GAL4. GFP fluorescence was observed in the tissues of GFP-positive animals, in which both promoters drove GAL4 gene expression. Animals that possessed only the GAL4 gene or UAS-GFP construct did not show GFP fluorescence. In addition, as a further test of the ability of the GAL4/UAS system to drive tissue-specific expression we constructed FiL-GAL4 lines with 3xP3-CFP as the transformation marker. FiL-GAL4 X UAS-GFP crosses showed GFP expression in the posterior silk gland, in which the endogenous FiL gene is normally expressed. These results show that the GAL4/UAS system is applicable to B. mori and emphasize the potential of this system for controlled analyses of B. mori gene function.

Author(s): Yuji Shiba (corresponding author) [1, 2]; Toshihito Gomibuchi [3]; Tatsuichiro Seto [3]; Yuko Wada [3]; Hajime Ichimura [3]; Yuki Tanaka [3]; Tatsuki Ogasawara [3]; Kenji Okada [3]; Naoko Shiba [...]

Dihydropyridine receptors (DHPRs), which are voltage-gated [Ca.sup.2+] channels, and ryanodine receptors (RyRs), which are intracellular [Ca.sup.2+] release channels, are expressed in diverse cell types, including skeletal and cardiac muscle. In skeletal muscle, there appears to be reciprocal signaling between the skeletal isoforms of both the DHPR and the RyR (RyR-1), such that [Ca.sup.2+] release activity of RyR-1 is controlled by the DHPR and [Ca.sup.2+] channel activity of the DHPR is controlled by RyR-1. Dyspedic skeletal muscle cells, which do not express RyR-1, lack excitation-contraction coupling and have an [approximately equal to]30-fold reduction in L-type [Ca.sup.2+] current density. Here we have examined the ability of the predominant cardiac and brain RyR isoform, RyR-2, to substitute for RyR-1 in interacting with the skeletal DHPR. When RyR-2 is expressed in dyspedic muscle cells, it gives rise to spontaneous intracellular [Ca.sup.2+] oscillations and supports [Ca.sup.2+] entry-induced [Ca.sup.2+] release. However, unlike RyR-1, the expressed RyR-2 does not increase the [Ca.sup.2+] channel activity of the DHPR, nor is the gating of RyR-2 controlled by the skeletal DHPR. Thus, the ability to participate in skeletal-type reciprocal signaling appears to be a unique feature of RyR-1.

The effect of ryanodine receptor (RyR-1) in dihydropyridine channel activity is determined using dyspedic mice myotubes as a destroyed RyR-1 gene. There was no excitation-contraction coupling recorded in dyspedic myotubes. L-type Ca2+-current density was reduced 30 times than the original. The application of of RyR-1 complementary DNA to dyspedic myotubes caused normal excitation-contraction coupling activity and normalized L-type Ca2+ currents. The enhanced L-type current was a result of the skeletal isoform of the dihydropyridine receptor (DHPR) as a calcium channel enhanced by a retrograde signal of RyR-1.

An analysis of the functions of amino-terminal portion S1-S3-S4 linker of the repeats of voltage-gated ion channels reveals that the difference in activation kinetics between cardiac and skeletal muscle L-type calcium channels derives from the amino acid composition of the S3 segment of repeat I and the linker linking S3 and S4 segments of repeat I. Activation can be transformed from gradual to fast even if a total of 11 conservative and 10 nonconservative amino acids change from skeletal muscle to cardiac dihydropyridine receptor sequence.