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

Long-term imaging of Ca²⁺ activity in spines in a cultured hippocampal pyramidal neuron.

<p> <b>A</b>, Z-projection of a representative CA3 pyramidal neuron expressing G-CaMP6-actin at 8 (upper) and 29 (lower) days <i>in vitro</i> (Div). After 7 days <i>in vitro</i>, the G-CaMP6-actin plasmid was introduced into the neuron via single-cell electroporation. Two spines of interest (S1, S2) are indicated by yellow circles. <b>B</b>, Changes in fluorescence at S1 and S2 upon supra-threshold electrical stimulation (Stim). The average spine Δ<i>F</i>/<i>F</i> ratios in response to supra-threshold stimulation were 253±30.5% and 201±46.6% at 8 Div and 29 Div, respectively (<i>n</i> = 25 spines, <i>P</i>>0.05, Student’s <i>t</i>-test).</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

Comparison of G-CaMP responses in acute cortical slices.

<p> <b>A</b>, Confocal image of G-CaMP6-expressing cortical pyramidal cells. The expression of G-CaMP6 was driven by the CAG promoter via <i>in utero</i> plasmid electroporation. Inset: Higher-magnification views are shown in the right panels. <b>B</b>, Representative Δ<i>F</i>/<i>F</i> traces in response to 1–4 spikes evoked at 50 Hz. <b>C</b>, Mean responses (Δ<i>F</i>/<i>F</i>) of G-CaMP3 and G-CaMP6 to spike trains. Error bars, s.e.m. (G-CaMP3, <i>n</i> = 4 cells; G-CaMP6, <i>n</i> = 5 cells).</p

Temperature dependence of G-CaMP6 signals.

<p> <b>A</b>, Representative traces of the fluorescence response (Δ<i>F</i>/<i>F</i>) of G-CaMP6 to a single spike at 25–28°C and at 37°C. <b>B</b>, Mean responses (Δ<i>F</i>/<i>F</i>) of G-CaMP6 to spike trains. Error bars, s.e.m. (<i>n</i> = 6 each). <b>C</b>, Rise and decay time constants of the responses of G-CaMP6 to single spikes. (*<i>P</i><0.05, paired <i>t</i>-test).</p

Electrophysiological properties of hippocampal neurons expressing G-CaMP6.

<p><b>A</b>, Left, input resistance. Middle, membrane capacitance. Right, resting potential. Error bars, s.e.m. (<i>n</i> = 6 each). There were no significant differences between the control and G-CaMP6 groups for any of the parameters (<i>P</i>>0.05, Student’s <i>t</i>-test). <b>B</b>, Left, spontaneous current under the voltage clamp at –70 mV. Middle, amplitude of the excitatory postsynaptic current. Right, frequency of the excitatory postsynaptic current. Error bars, s.e.m. (<i>n</i> = 6 each, <i>P</i>>0.05, Student’s <i>t</i>-test).</p

Ca²⁺ imaging of cholinergic DA motoneurons in freely moving <i>C. elegans</i>.

<p> <b>A</b>, Confocal images of L1 larvae expressing G-CaMP6 (<i>jqEx97</i>) or G-CaMP3 (<i>jqEx216</i>) in the DA motoneurons. In both transgenic strains, DsRed-Express-1 is co-expressed in the DA motoneurons. TL, transmitted-light image. Arrows indicate the DA7 motoneuron analyzed in <b>B</b>. <b>B,</b> Representative spontaneous fluorescence responses (Δ<i>R</i>/<i>R</i>) of G-CaMPs from DA7 cholinergic neurons in transgenic worms during locomotion. <b>C</b>, Mean peak responses (Δ<i>R</i>/<i>R</i>). Error bars, s.e.m. (<i>n</i> = 10 each from 4 worms, *<i>P</i> = 0.0020, Student’s <i>t</i>-test). Movies of the recordings are available as supplementary information (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051286#pone.0051286.s001" target="_blank">Movies S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051286#pone.0051286.s002" target="_blank">S2</a>).</p

Characterization of G-CaMPs in cultured hippocampal slices.

<p> <b>A</b>, Expression of G-CaMP6 in hippocampal CA3 pyramidal neurons. Inset: Higher-magnification views are shown in the bottom panels. <b>B</b>, Baseline fluorescence of hippocampal neurons expressing G-CaMP3, G-CaMP6 and G-CaMP8. No significant differences in variance were detected among the three groups (<i>P</i>>0.05, χ² = 2.90, Bartlett’s test). Error bars, s.d. (<i>n</i> = 7 each, <i>P</i>>0.05, Tukey’s test). <b>C</b>, Representative traces of the response (Δ<i>F</i>/<i>F</i>) to spike trains. The frequency of stimuli was 50 Hz. Right: Magnified views of single spikes. <b>D</b>, Mean responses (Δ<i>F</i>/<i>F</i>) and SNRs of G-CaMP3 (black), G-CaMP6 (red) and G-CaMP8 (blue). Inset: Magnified views of 1–2 spikes. Error bars, s.e.m. (<i>n</i> = 7 each). <b>E</b>, Rise and decay time constants for the responses to single spikes. Error bars, s.e.m. (<i>n</i> = 7 each; *<i>P</i><0.05 in Tukey’s post-hoc test following one-way ANOVA). <b>F</b>, Trial-averaged responses of G-CaMP6 to spike trains. Gray, individual traces (<i>n</i> = 10 trials); red, averaged traces. Bars indicate stimulus timing. Inset: Magnified views.</p

Characterization of G-CaMPs <i>in vitro</i> and in HeLa cells.

<p> <b>A</b>, Schematic representation. Mutations are indicated with respect to G-CaMP2. RSET and M13 are tags that encode hexahistidine and a target peptide for Ca²⁺-bound CaM derived from MLCK, respectively. The amino-acid numbers of EGFP and CaM are indicated in parentheses. <b>B</b>, Dynamic range (<i>F</i>_max/<i>F</i>_min) and Ca²⁺ affinity (<i>K</i>_d). Error bars, s.d. (<i>n</i> = 3 each). <b>C</b>, Ca²⁺ titration curve. Curves were fit according to the Hill equation. <i>K</i>_d is shown in <b>B</b>. <b>D</b>, Normalized fluorescence and absorbance (inset) spectra of G-CaMP6 and G-CaMP8 in 1 µM Ca²⁺ or 1 mM EGTA. <b>E</b>, Fluorescence images of HeLa cells expressing G-CaMPs. Bars, 30 µm. <b>F</b>, Time course of the changes (Δ<i>F</i>/<i>F</i>) in G-CaMP fluorescence in response to 100 µM ATP. Error bars, s.d. <b>G</b>, Baseline fluorescence and peak responses (Δ<i>F</i>/<i>F</i>) to ATP application in HeLa cells.</p