Additional file 1: of Role of tyramine in calcium dynamics of GABAergic neurons and escape behavior in Caenorhabditis elegans

Figure S1. ICaST system for optogenetic control and simultaneous calcium imaging in freely moving animals. The light paths are indicated by colored arrows. Details are described in the Materials and Methods and a previous report [27]. Figure S2. R-CaMP2 imaging in RME. (A) Representative images of a transgenic animal expressing both R-CaMP2 and EGFP in RME neurons in forward (top panels) and backward (bottom panels) movements. Transmitted-light images (TD), raw fluorescent images of R-CaMP2 and EGFP, fluorescent merged images, and pseudocolor ratio images (R-CaMP2/EGFP) are shown. (B) Fluorescent intensity ratio values (R=R-CaMP2/EGFP) of RME in a freely moving animal are plotted as a function of time. (C) Quantitative analysis of mean fluorescent ratio changes of RME during forward (gray) and backward (red) locomotion. The mean value of R during forward locomotion was normalized as 100%. Figure S3. tbh-1 mutants exhibit normal calcium responses in RME during backward locomotion. A representative calcium trace of RME in tbh-1(ok1193) mutants. Figure S4. tph-1 and cat-2 mutants exhibit normal calcium responses in RME during backward locomotion. (A) Biosynthetic pathways of serotonin and dopamine. Genes encoding synthetic enzymes are shown under the arrows. (B, C) Calcium dynamics of RME in tph-1 (mg280) (B) and cat-2 (jq6) (C) mutants during spontaneous locomotion. Table S1. C. elegans strains used in this study. Table S2. Transgenic lines generated in this study. Table S3. Mutations and primers for genotyping. Table S4. Primers for molecular biology. (PDF 359 kb

Generation and Imaging of Transgenic Mice that Express G-CaMP7 under a Tetracycline Response Element

<div><p>The spatiotemporally controlled expression of G-CaMP fluorescent calcium indicator proteins can facilitate reliable imaging of brain circuit activity. Here, we generated a transgenic mouse line that expresses G-CaMP7 under a tetracycline response element. When crossed with a forebrain-specific tetracycline-controlled transactivator driver line, the mice expressed G-CaMP7 in defined cell populations in a tetracycline-controlled manner, notably in pyramidal neurons in layer 2/3 of the cortex and in the CA1 area of the hippocampus; this expression allowed for imaging of the <i>in vivo</i> activity of these circuits. This mouse line thus provides a useful genetic tool for controlled G-CaMP expression <i>in vivo</i>.</p></div

Transgene expression in brain areas outside the neocortex and hippocampus.

<p>The expression patterns of G-CaMP7 and DsRed2 in coronal sections of the olfactory bulb, piriform cortex, amygdala and striatum from TRE-G-CaMP7 x CaMKII-tTA mice are shown at lower (left panels for each brain area) and higher (right panels) magnifications. Arrowheads indicate olfactory glomeruli intensely labeled by G-CaMP7. Arrows represent examples of striatal neurons strongly expressing G-CaMP7. Gl, glomerular layer; EP, external plexiform layer; MC, mitral cell layer; IP, internal plexiform layer; Gr, granule cell layer; I, II, and III, layers I, II, and III of the piriform cortex, respectively; LA, lateral amygdala; BLA, basolateral amygdala; St, striatum; EC, external capsule; D, dorsal; L, lateral; M, medial. Scale bar = 100 μm.</p

Two-photon imaging of spontaneous cortical layer 2/3 circuit activity in anesthetized TRE-G-CaMP7 x CaMKII-tTA mice at 4 months of age.

<p><b>A</b>, Neurons expressing G-CaMP7 (left) and DsRed2 (right) were imaged in the posterior cortex 60, 200, and 350 μm from the pial surface. The darker areas in each image are shadows of vessels. Scale bar = 100 μm. <b>B</b>, The positions and numbers of 13 active neurons are indicated in an average G-CaMP7 fluorescence image acquired at a depth of 200 μm (left). Similarly, those of 6 neuropil areas are shown in the same average G-CaMP7 fluorescence image (right). <b>C</b>, Baseline-normalized G-CaMP7 fluorescence time traces for the same 13 cells (top) and 6 neuropil regions (bottom left) and baseline-normalized DsRed2 fluorescence time traces for the same 6 neuropil regions (bottom right). <b>D</b>, A cross-correlation matrix of G-CaMP7 fluorescence time traces of the 13 cells. <b>E</b>, Example time-lapse images of G-CaMP7 fluorescence during spontaneous network activity in layer 2/3 of the cortex. Cell 2 and Cell 13 (as designated in <b>B</b>) were synchronously activated multiple times. Time stamps are indicated above the images. Active cells are indicated by red arrows.</p

Visualization 1

Dorsal hippocampal CA1 area of the transgenic mouse imaged using the microendoscope (Fig.5(b)). Overlaid images of G-CaMP7 (green) and DsRed2 (red) fluorescence signals are shown. The images were acquired 0 to 200 μm from the hippocampal surface at 5-μm intervals. Depths are indicated in the upper left corner of the movie

Visualization 4

Synchronous network activity in the amygdala imaged using the fast varifocal microendoscope (Fig. 6(c)). Images acquired quasi-simultaneously at different depths are shown in top (0 V) and bottom (0.35 V, 120 μm below) panels. The movie is spatially binned by two to reduce the file size. The playback speed is 5x faster than the speed of real activity. Time stamps are indicated in the upper left corner of each panel

Two-photon imaging of spontaneous hippocampal CA1 circuit activity in anesthetized TRE-G-CaMP7 x CaMKII-tTA mice at 4 months of age.

<p><b>A</b>, Different subcellular compartments of pyramidal neurons expressing G-CaMP7 (left) and DsRed2 (right) were imaged in the dorsal CA1 hippocampus 50, 95 and 140 μm from the hippocampal surface. Scale bar = 50 μm. <b>B</b>, The positions and numbers of 12 active neurons selected for analysis are indicated in an average G-CaMP7 fluorescence image acquired at a depth of 145 μm. <b>C</b>, Baseline-normalized G-CaMP7 fluorescence time traces of the 12 active cells. <b>D</b>, A cross-correlation matrix of G-CaMP7 fluorescence time traces of the 12 active cells. <b>E</b>, Example time-lapse images of G-CaMP7 fluorescence during spontaneous network activity in the CA1 hippocampus. Cells 7 and 2 (as designated in <b>B</b>) were sequentially activated multiple times. Only two out of nine total sequential activation events are shown here. Time stamps are indicated at the bottom of the images. Δt represents the image sampling interval (0.43 s). Active cells 7 and 2 are indicated by red arrows.</p

Reduced incorporation of exogenous C26:0 fatty acid into sphingomyelin in <i>acs-20</i> and <i>acs-22</i> mutants.

<p>Incorporation of [¹⁴C]C26:0, [¹⁴C]C16:0 and [¹⁴C]C20:4 into sphingomyelin is expressed as the percentage of the wild-type value. All experiments were performed at 20°C. Bars represent SEMs of two independent experiments.</p

<i>acs-20</i> mutants are strongly stained with lipophilic dyes, but their lipid contents are not altered.

<p>(A) Wild-type (left panels) and <i>acs-20 (tm3232)</i> mutant (right panels) animals are stained with Nile Red, C1-Bodipy-C12, and Sudan black, respectively. Scale bars  = 100 µm. (B) Amounts of triacylglycerol in wild-type and <i>acs-20</i> mutant animals. (C) Lipid composition in wild-type and <i>acs-20</i> mutant animals. (B, C) Error bars indicate SEM of three measurements.</p

Genetically Encoded Green Fluorescent Ca²⁺ Indicators with Improved Detectability for Neuronal Ca²⁺ Signals

<div><p>Imaging the activities of individual neurons with genetically encoded Ca²⁺ indicators (GECIs) is a promising method for understanding neuronal network functions. Here, we report GECIs with improved neuronal Ca²⁺ signal detectability, termed G-CaMP6 and G-CaMP8. Compared to a series of existing G-CaMPs, G-CaMP6 showed fairly high sensitivity and rapid kinetics, both of which are suitable properties for detecting subtle and fast neuronal activities. G-CaMP8 showed a greater signal (<em>F</em>_max/<em>F</em>_min = 38) than G-CaMP6 and demonstrated kinetics similar to those of G-CaMP6. Both GECIs could detect individual spikes from pyramidal neurons of cultured hippocampal slices or acute cortical slices with 100% detection rates, demonstrating their superior performance to existing GECIs. Because G-CaMP6 showed a higher sensitivity and brighter baseline fluorescence than G-CaMP8 in a cellular environment, we applied G-CaMP6 for Ca²⁺ imaging of dendritic spines, the putative postsynaptic sites. By expressing a G-CaMP6-actin fusion protein for the spines in hippocampal CA3 pyramidal neurons and electrically stimulating the granule cells of the dentate gyrus, which innervate CA3 pyramidal neurons, we found that sub-threshold stimulation triggered small Ca²⁺ responses in a limited number of spines with a low response rate in active spines, whereas supra-threshold stimulation triggered large fluorescence responses in virtually all of the spines with a 100% activity rate.</p> </div