deletions | additions       diff --git a/Methods_electrophys.tex b/Methods_electrophys.tex  index 2dd4bc2..4d54be6 100644  --- a/Methods_electrophys.tex  +++ b/Methods_electrophys.tex 
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Data for tail currents were filtered at 10 kHz and digitized at 20 kHz.   Peak currents and exponential fits to currents were analyzed using Clampfit software (Axon instruments, Foster City, CA, USA).   Activation and inactivation time constants of T-type channel currents elicited by step pulses were estimated by fitting individual current traces with a double exponential function: $A1(1-exp(-t/\tau1)) + A2(1-exp(-t/\tau2))$ where $A1$ and $A2$ are the coefficients for the activation and inactivation exponentials, $t$ is time, and $\tau1$ and $\tau2$ are the activation and inactivation time constants, respectively.  The smooth curves for channel activation and steady-state inactivation were from fitting data with a Boltzmann equation: $\frac{1}{1+exp[(V_{50}-V)/S_{act}]}$ , $1/\{1+exp[(V_{50}-V)/S_{act}]\}$,  where $V_{50}$ is the potential for half-maximal activation and $S_{act}$ is the slope conductance. Dose-response curves for Ni\textsuperscript{2+} inhibition of T-type channel currents were derived by fitting the data using a Hill equation: $B = 1/(1 + \textrm{IC}_{50}/[\textrm{Ni}^{2+}]^n)$, where $B$ is the normalized block, $\textrm{IC}_{50}$ is the concentration of $\textrm{Ni}^{2+}$ giving half maximal blockade, and $n$ is the Hill coefficient.   
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        diff --git a/Methods_sleep.tex b/Methods_sleep.tex  index e60e632..c435e73 100644  --- a/Methods_sleep.tex  +++ b/Methods_sleep.tex 
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For sleep analysis, 3-4 day-old female flies were placed individually into 65 mm X 5mm glass tubes with one end filled with 2\% agar/5\% sucrose food and the other end plugged with cotton.  Periods of activity were defined as periods with a beam break frequency higher than 1 per minute, and periods of sleep were defined as periods with a beam break frequency lower than 1 per 5 minutes\cite{Shaw:2000ui}.  After one day of habituation in an incubator (25$\,^{\circ}\mathrm{C}$, 60\% humidity), sleep during two days of 12hr:12hr light-dark cycle and following two days of dark period was analysed using Counting Macro \cite{pfeiffenberger:2010ab}.  For experiment using elav-GeneSwitch(GS)-gal4, flies were maintained on normal food containing 500 $\mu$M RU486 (M8046, Sigma-Aldrich) dissolved in ethanol(1\%) for two days before experiment.  Control flies were maintained on normal food containing only ethanol (1\%).  During the experiment, flies were placed in 2\% agar/5\% sucrose food with or without 500 $\mu$M RU486.   For  circadian locomotor analysis, 1-3 day-old male flies were used. Activity was measured every 30 min bin and analysed using ClockLab analysis software (Actimetrics) and Counting Macro\cite{pfeiffenberger:2010aa}. Macro software.  Significance level of the $\chi$\textsuperscript{2} periodogram was set to $\alpha$ = 0.05. Flies with a power of significance (P-S) $\geq$10 were considered as rhythmic.  diff --git a/Results_expression.tex b/Results_expression.tex  index db5ff8f..232edb9 100644  --- a/Results_expression.tex  +++ b/Results_expression.tex 
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This produced the \emph{GFP::DmCa\textsubscript{v}3} line, which expresses an N-terminally GFP-tagged DmCa\textsubscript{v}3 under the control of its own endogenous promoter.  In these \emph{GFP::DmCa\textsubscript{v}3} flies, GFP fluorescence appears broadly across the brain (Fig. \ref{fig:2}b).  GFP::DmCa\textsubscript{v}3 \emph{GFP::DmCa\textsubscript{v}3}  is expressed in well-structured neuropils like the antennal lobes, the mushroom bodies, the central complex (Fig. \ref{fig:2}c-h), the optic lobes, as well as in some of the less-structured neuropils. The central complex---comprising the fan-shaped body, ellipsoid body, noduli, and protocerebral bridge---shows the strongest expression with the ventral fan-shaped body and ventral noduli particularly prominent (Fig. \ref{fig:2}e and g).  In mushroom body neurons, there is far more GFP::DmCa\textsubscript{v}3 \emph{GFP::DmCa\textsubscript{v}3}  in the dendrite-rich calyx of the dorso-posterior brain (Fig. \ref{fig:2}h) than the axon-rich lobes of the anterior brain (Fig. \ref{fig:2}d). GFP::DmCa\textsubscript{v}3 \emph{GFP::DmCa\textsubscript{v}3}  is also limited to the posterior mushroom body peduncles, which are the fiber tracks that join the posterior calyces with the anterior mushroom body lobes (Fig. \ref{fig:2}f). These results suggest strict regulation of the subcellular localization of DmCa\textsubscript{v}3 channels in the brain.  We next visualized the projections of DmCa\textsubscript{v}3-expressing neurons using another knock-in allele, \emph{DmCa\textsubscript{v}3\textsuperscript{Gal4}}.  In \emph{DmCa\textsubscript{v}3\textsuperscript{Gal4}}, the first coding exon and flanking introns of DmCa\textsubscript{v}3 are replaced by the Gal4 coding sequence.  This puts GAL4 expression under the control of the endogenous DmCa\textsubscript{v}3 promoter (Fig. \ref{fig:3}a).  Consistent with our results using GFP::DmCa\textsubscript{v}3, \emph{GFP::DmCa\textsubscript{v}3},  \emph{DmCa\textsubscript{v}3\textsuperscript{Gal4}} drives the expression of a membrane-tethered mCherry (UAS-mChRFP) (\emph{UAS-mCD8-mChRFP})  broadly across the brain (Fig. \ref{fig:S1}a). The DmCa\textsubscript{v}3\textsuperscript{Gal4}}>mCherry \emph{DmCa\textsubscript{v}3\textsuperscript{Gal4}\textgreater{}mCherry}  and GFP::DmCa\textsubscript{v}3 \emph{GFP::DmCa\textsubscript{v}3}  signals are strongly co-localized, including in the central complex and mushroom bodies (\ref{fig:S1}b and c}). This suggests both reagents reflect proper expression from the same endogenous DmCa\textsubscript{v}3 promoter.  diff --git a/Results_knockdown.tex b/Results_knockdown.tex  index 999b167..d857bf5 100644  --- a/Results_knockdown.tex  +++ b/Results_knockdown.tex 
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We examined whether neuronal T-type channel is responsible for increased sleep of DmCa\textsubscript{v}3 mutants.  Pan-neuronal knockdown of DmCa\textsubscript{v}3 using elav-gal4 driven DmCa\textsubscript{v}3 RNAi expression increased sleep both LD and DD, showing neuronal T-type Ca\textsuperscript{2+} channel is responsible for sleep regulation (Fig \ref{fig:5}).  Knockdown of DmCa\textsubscript{v}3 with \emph{DmCa\textsubscript{v}3\textsuperscript{Gal4}} driver also showed increased sleep after third day of continuous dark phase ensuring that gal4 expression of \emph{DmCa\textsubscript{v}3\textsuperscript{Gal4}} represents endogenous DmCa\textsubscript{v}3 expression (Fig. \ref{fig:S3}). We also examined whether adult stage-specific knock-down of DmCa\textsubscript{v}3 induces increased sleep.  DmCa\textsubscript{v}3 knock-down by drug-inducible pan-neuronal gal4 driver (\emph{Elav-GeneSwitch(GS)-gal4}) increased sleep in DD compared to drug unfed control (Fig.\ref{fig:S}), suggesting sleep phenotype of DmCa\textsubscript{v}3 mutants are not due to the developmental defect.  Next, we wondered if any specific brain area is responsible for sleep regulating role of DmCa\textsubscript{v}3.  We knock downed DmCa\textsubscript{v}3 using broad neuronal gal4 drivers as well as specific gal4 drivers that cover previously known sleep regulating centers.  None of these gal4-driven knock-down of DmCa\textsubscript{v}3 induced sleep increase (Fig. \ref{fig:S4}), suggesting DmCa\textsubscript{v}3 may function in previously unknown circuits or function in more broad area that any specific gal4 lines used did not cover.         diff --git a/figures/Fig_5/caption.tex b/figures/Fig_5/caption.tex  index b1a61c5..01ceb79 100644  --- a/figures/Fig_5/caption.tex  +++ b/figures/Fig_5/caption.tex 
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\label{fig:5}  \textbf{Pan-neuronal knock-down of DmCa\textsubscript{v}3 increases sleep.}  \\  \textbf{(a)} Sleep profiles of over two days of 12h:12h light-dark cycle (LD) and two days of continuous dark condition (DD). Pan-neuronal gal4 driven DmCa\textsubscript{v}3 RNAi flies (\emph{Elav$>$RNAi}, (\emph{Elav\textgreater{}RNAi},  orange line, n=44) have increased sleep compared to both gal4 control(\emph{elav-gal4/+}, black line, n=38) and UAS control (\emph{UAS-DmCa\textsubscript{v}3 RNAi/+}, grey line, n=42). Sleep in 30 minute intervals was plotted.  White, black and grey bar denote light phase, dark phase and subjective light phase, respectively.  ZT, zeitgeber time. 
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        diff --git a/figures/Fig_S/Fig_S.png b/figures/Fig_S/Fig_S.png  new file mode 100644  index 0000000..179e63b  Binary files /dev/null and b/figures/Fig_S/Fig_S.png differ diff --git a/figures/Fig_S/caption.tex b/figures/Fig_S/caption.tex  new file mode 100644  index 0000000..b01855c  --- /dev/null  +++ b/figures/Fig_S/caption.tex 
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\label{fig:S}  \textbf{Adult stage-specific knock-down of DmCa\textsubscript{v}3 increases sleep in constant darkness.}  \\  Sleep during subjective daytime in continuous dark period (DD).   Flies fed RU486 in adult stage (\emph{Elav-GS\textgreater{}RNAi}, black) show significantly increased sleep in second day of DD compared to control flies (\emph{Elav-GS\textgreater{}RNAi}, white).  Data are represented as mean $\pm$ s.e.m.  For statistical analysis, One-way ANOVA followed by Tukey-HSD post hoc test was performed.  *p$<$0.05.  diff --git a/figures/Fig_S/size.tex b/figures/Fig_S/size.tex  new file mode 100644  index 0000000..2fcf78b  --- /dev/null  +++ b/figures/Fig_S/size.tex 
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height = 700\nwidth = 500  diff --git a/figures/Fig_S3/caption.tex b/figures/Fig_S3/caption.tex  index dec358d..a24471f 100644  --- a/figures/Fig_S3/caption.tex  +++ b/figures/Fig_S3/caption.tex 
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\label{fig:S3}  \textbf{\emph{DmCa\textsubscript{v}3\textsuperscript{Gal4}}-driven knock-down of DmCa\textsubscript{v}3 increases sleep in constant darkness.}  \\  \textbf{(a)} Sleep profiles of \emph{DmCa\textsubscript{v}3\textsuperscript{Gal4}/+} (black line, n=63), \emph{UAS-DmCa\textsubscript{v}3-RNAi/+} (grey line, n=63) and \emph{DmCa\textsubscript{v}3\textsuperscript{Gal4}$>$DmCa\textsubscript{v}3-RNAi} \emph{DmCa\textsubscript{v}3\textsuperscript{Gal4}\textgreater{}DmCa\textsubscript{v}3-RNAi}  (orange line, n=59) over two days of 12h:12h light-dark cycle (LD) and four days of continuous dark condition (DD). Sleep in 30 minute intervals was plotted.  White, black and grey bar denote light phase, dark phase and subjective light phase, respectively.  ZT, zeitgeber time. 
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        diff --git a/figures/Fig_S4/caption.tex b/figures/Fig_S4/caption.tex  index a41b239..5e0c8ca 100644  --- a/figures/Fig_S4/caption.tex  +++ b/figures/Fig_S4/caption.tex 
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\\  \textbf {(a)} Averaged sleep amount over two days of 12h:12h light-dark cycle (LD).  \textbf {(b)} Averaged sleep amount over two days of continuous dark condition (DD).  White, grey, black bar denotes RNAi control, Gal4 control and Gal4 driven RNAi(Gal4$>$RNAi), RNAi(Gal4\textgreater{}RNAi),  respectively (n=21-83). PI, pars intercerebralis, MB, mushroom body.   Data are represented as mean $\pm$ s.e.m.  For statistical analysis, Welch's ANOVA followed by Games-Howell post hoc test was performed.  *p$<$0.05, **p$<$0.01, ***p$<$0.001.        diff --git a/layout.md b/layout.md  index 1bd4b6c..3ea1692 100644  --- a/layout.md  +++ b/layout.md 
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figures/Fig_S1/Fig_S1.png  figures/Fig_S2/Fig_S2.png  figures/Fig_S3/Fig_S3.png  figures/Fig_S/Fig_S.png  figures/Fig_S4/Fig_S4.png