deletions | additions       diff --git a/Methods_antibody.tex b/Methods_antibody.tex  index e3eed19..21e510b 100644  --- a/Methods_antibody.tex  +++ b/Methods_antibody.tex 
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\subsection*{Antibody generation and western blotting}  A polyclonal antisera against DmCa\textsubscript{v}3 was generated using antigen derived from the 302 C-terminal amino acids of DmCa\textsubscript{v}3 (cloning primers: 5'-GAATTCCAAATTAATCCAATCCGTA-3', 5'-GCGGCCGCTTAGTCCATGGAGGATT-3'). \seqsplit{5'-GAATTCCAAATTAATCCAATCCGTA-3'}, \seqsplit{5'-GCGGCCGCTTAGTCCATGGAGGATT-3'}).  His-tagged antigen was expressed in \emph{E. coli}, purified and injected into rabbits to generate an immune response (YoungIn Frontier, South Korea).  Western blot analyses were performed according to standard protocols using rabbit antisera obtained after the third DmCa\textsubscript{v}3 antigen boost.  $\beta$-Actin-specific antibodies (Santa Cruz Biotechnology, sc-47778) were used for the loading control. diff --git a/Methods_chemicals.tex b/Methods_chemicals.tex  index cce334a..9ed8669 100644  --- a/Methods_chemicals.tex  +++ b/Methods_chemicals.tex 
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Most of chemicals for electrophysiological recordings were purchased from Sigma-Aldrich (St. Louis, MO, USA).  A 100 mM nickel-chloride stock solution was made in deionized water.  A series of nickel solutions (in $\mu$M: 0.3, 1, 3, 10, 30, 100, 300, 1000, and 3000) were prepared by diluting the stock solution with 10 mM Ba\textsuperscript{2+} recording solution (in mM: 10 BaOH\textsubscript{2}, 90 NaOH, 1 KOH, 5 HEPES, pH 7.4 adjusted with methanesulfonic acid) before every nickel inhibition experiment.       diff --git a/Methods_cloning.tex b/Methods_cloning.tex  index 57be701..4996abd 100644  --- a/Methods_cloning.tex  +++ b/Methods_cloning.tex 
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We generated a full-length DmCa\textsubscript{v}3 (CG15899) cDNA by piecemeal PCR amplification.  Total RNA extracted from adult heads using Trizol reagents (Invitrogen) was reverse transcribed using RevertAid First Strand cDNA Synthesis Kit (Fermentas).  Six adjacent DNA fragments that cover the entire DmCa\textsubscript{v}3 cDNA were obtained by PCR amplification.   Primer sets were designed based on the FlyBase (FB2011_07) (FB2011\_07)  annotation for DmCa\textsubscript{v}3. Hind III and Xba I sites were inserted at the 5' end of fragment 1 and 3' end of fragment 6, respectively.  Primer sets: fragment 1 (5'-CGAGATAAGCTTAAAATGCTGCCACAGCCA-3', 5'-GCATCAGACTACATCGCTGTC-3'), (\seqsplit{5'-CGAGATAAGCTTAAAATGCTGCCACAGCCA-3'}, \seqsplit{5'-GCATCAGACTACATCGCTGTC-3'}),  fragment 2 (5'-CTGGACACGCTGCCCATGCTG-3', 5'-TTCCAGCTCCTCCACTTGCAC-3'), (\seqsplit{5'-CTGGACACGCTGCCCATGCTG-3'}, \seqsplit{5'-TTCCAGCTCCTCCACTTGCAC-3'}),  fragment 3 (5'-CAACGGTGGCTCCAACAGTCG-3', 5'-CCACTGGCGGAAGCTCATGCC-3'), (\seqsplit{5'-CAACGGTGGCTCCAACAGTCG-3'}, \seqsplit{5'-CCACTGGCGGAAGCTCATGCC-3'}),  fragment 4 (5'-GCCACGCCTCTCCAAGATCCG-3', 5'-GACGATAAGAGCGTTTGCACG-3'), (\seqsplit{5'-GCCACGCCTCTCCAAGATCCG-3'}, \seqsplit{5'-GACGATAAGAGCGTTTGCACG-3'}),  fragment 5 (5'-TCTGAAACTAGTCGTGCAAAC-3', 5'-TGGAAGTACTGGACGGTCTGC-3'), (\seqsplit{5'-TCTGAAACTAGTCGTGCAAAC-3'}, \seqsplit{5'-TGGAAGTACTGGACGGTCTGC-3'}),  and fragment 6 (5'-AATCCCAGCCTGACCAGCTCG-3', 5'-TCTAGATTAGTCCATGGAGGATTGGGGTGA-3'). (\seqsplit{5'-AATCCCAGCCTGACCAGCTCG-3'}, \seqsplit{5'-TCTAGATTAGTCCATGGAGGATTGGGGTGA-3'}).  Amplified PCR fragments were sequenced and assembled into pBlueScript II KS (+) using sequential restriction enzyme digests.  Clones 2 and 3 contained isoform-specific exons.  Of the combinations that were amplified by PCR, we chose to proceed to assembling the RB and RC isoforms.  We observed frequent, but inconsistent mutations and instances of A to G RNA editing in fragments 3 and 5.  To achieve a final DmCa\textsubscript{v}3 cDNA matching the FlyBase annotation, we reverted one edited site in fragment 3 (5'-AGTTCAGAGC-3') (\seqsplit{5'-AGTTCAGAGC-3'})  by site-directed mutagenesis. Since fragment 5 had so many inconsistencies and contained no introns, we used genomic DNA as a template for fragment 5 instead of cDNA.  The final assembled full-length cDNAs were cut with HindIII / XbaI and subcloned into pcDNA3-HE3 downstream of the 5'-UTR from the \emph{Xenopus laevis} $\beta$-globin gene to improve expression in \emph{Xenopus} oocytes. diff --git a/Methods_fly_stocks.tex b/Methods_fly_stocks.tex  index b8f48ff..bb46412 100644  --- a/Methods_fly_stocks.tex  +++ b/Methods_fly_stocks.tex 
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EP line G1047 was obtained from \href{http://genexel.kaist.ac.kr}{Genexel}.  \emph{c465-Gal4} and \emph{210y-Gal4} were gifts from J. Douglas Armstrong\cite{Young:2010jq}.  The following stocks were all described previously: \emph{elav-Gal4}\cite{Lin:1994vn}, \emph{elav-GS-Gal4}\cite{Osterwalder:2001cl}, \emph{Cha-Gal4}\cite{Kitamoto:2001ue} \emph{c161-Gal4}\cite{renn:1999aa}, \emph{104y-Gal4}\cite{sakai:2006aa}, \emph{c309-Gal4}\cite{connolly:1996aa}, \emph{MB247-Gal4}\cite{zars:2000aa}, \emph{pdf-Gal4}\cite{renn:1999ab}, \emph{TH-Gal4}\cite{friggi-grelin:2003aa}, \emph{Tdc2-Gal4}\cite{alekseyenko:2010aa}, \emph{c232-Gal4}\cite{renn:1999aa}, \emph{TRH-Gal4}\cite{alekseyenko:2010aa}, \emph{GMR-Gal4}\cite{freeman:1996aa}, \emph{c929-Gal4}\cite{taghert:2001aa}, \emph{clk8.0-Gal4}\cite{glossop:2003aa} and \emph{dilp2-Gal4}\cite{Rulifson:2002cg}.            diff --git a/Methods_knockin_alleles.tex b/Methods_knockin_alleles.tex  index 52b7661..3d5f830 100644  --- a/Methods_knockin_alleles.tex  +++ b/Methods_knockin_alleles.tex 
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\subsection*{Generation of knock-in alleles}  5' and 3' homologous arms surrounding the \emph{DmCa\textsubscript{v}3} locus were PCR amplified using \emph{w\textsuperscript{1118}} genomic DNA with the following primers: 5'-CGAGATGAATTCTAGCCTCATCAACTGAGC-3', 5'-CGAGATGCGGCCGCGAGCAAGCACTAATAGCA-3', 5'-GAGATACTAGTCATGCTACAATGTCAGCA-3', 5'-CGAGATCTCGAGGGCCACGTATAGGGATGC-3'. 5'-CGAGATGAATTCTAGCCTCATCAACTGAGC-3'}, \seqsplit{5'-CGAGATGCGGCCGCGAGCAAGCACTAATAGCA-3'}, \seqsplit{5'-GAGATACTAGTCATGCTACAATGTCAGCA-3'}, \seqsplit{5'-CGAGATCTCGAGGGCCACGTATAGGGATGC-3'}.  The homologous arms were then inserted into the \emph{pGX-attP} vector (\href{https://dgrc.bio.indiana.edu/product/View?product=1293}{DGRC \#1293}).  P\{Donor\} flies were generated by P-element based transgenesis of \emph{pGX-attP} containing the homologous arms into the \emph{w\textsuperscript{1118}} genetic background (Genetic Services, Inc., US) and crossed to Flp I-Sce I flies for homologous recombination.  Candidate for proper targeting (i.e., flies with red or mosaic eyes) were selected and verified by PCR.  The \emph{white} marker was removed from a verified strain via Cre-mediated recombination.  The resulting \emph{white}\textsuperscript{-} line was used as a founder (\emph{DmCa\textsubscript{v}3\textsuperscript{Founder,w-}}) for site-specific DNA integration.  \emph{DmCa\textsubscript{v}3\textsuperscript{Gal4}}, \emph{DmCa\textsubscript{v}3\textsuperscript{Rescue}} and \emph{GFP::DmCa\textsubscript{v}3} lines were generated by $\phi$C31 integrase-mediated site-specific integration.   The Gal4 insert (i.e., splice acceptor-Gal4 CDS-poly A) was amplified from the \emph{pBS-KS-attB1-2-GT-SA-GAL4-Hsp70pA} vector (\href{https://dgrc.bio.indiana.edu/product/View?product=1325}{DGRC \#1325}) with the following primers: 5'-CGTACTCCACGAATTTCTAGAAGTCGATCCAACAT-3' \seqsplit{5'-CGTACTCCACGAATTTCTAGAAGTCGATCCAACAT-3'}  and 5'-ACCGGCGCGCCTCGACTCTAGAACTAGTGGATCTA-3'. \seqsplit{5'-ACCGGCGCGCCTCGACTCTAGAACTAGTGGATCTA-3'}.  The resulting amplified DNA fragment was sequenced and inserted into the \emph{pGE-attB\textsuperscript{GMR}} vector (\href{https://dgrc.bio.indiana.edu/product/View?product=1295}{DGRC \#1295}) using the EZ-FusionTM cloning kit (Enzynomics, South Korea).   The Rescue insert was PCR amplified from \emph{w\textsuperscript{1118}} genomic DNA with the following primers: 5'-GCAGAATTCAATCGATTCCATAGATCCGC-3' \seqsplit{5'-GCAGAATTCAATCGATTCCATAGATCCGC-3'}  and 5'-GCACTCGAGAATTTTGCAACAGGCAGCTA-3'. \seqsplit{5'-GCACTCGAGAATTTTGCAACAGGCAGCTA-3'}.  The resulting fragment was inserted into the EcoR I/Xho I site of the \emph{pGE-attB\textsuperscript{GMR}} vector.   The GFP insert along with a (Gly-Gly-Ser)x4 linker was amplified from the \emph{pBS-KS-attB1-2-PT-SA-SD-1-EGFP-FIAsH-StrepII-TEV-3xFlag} vector (\href{https://dgrc.bio.indiana.edu/product/View?product=1306}{DGRC \#1306}) with the following primers: 5'-GCACCCCAGAAAATGGTGTCCAAGGGCGAGGAGCT-3' \seqsplit{5'-GCACCCCAGAAAATGGTGTCCAAGGGCGAGGAGCT-3'}  and 5'-CGCTGGCTGTGGCAGGGAACCTCCGCTTCCACCGC-3'. \seqsplit{5'-CGCTGGCTGTGGCAGGGAACCTCCGCTTCCACCGC-3'}.  The resulting fragment was inserted downstream of the ATG start site in the Rescue construct by inverse PCR (5'-CTGCCACAGCCAGCGGCAGCG-3', 5'-CATTTTCTGGGGTGCCAACTA-3') (\seqsplit{5'-CTGCCACAGCCAGCGGCAGCG-3'}, \seqsplit{5'-CATTTTCTGGGGTGCCAACTA-3'})  using the 5X In-Fusion HD Enzyme Premix (Clontech). \emph{pGE-attB\textsuperscript{GMR}} vectors containing the Gal4, Rescue, and GFP-tagging constructs were injected into \emph{DmCa\textsubscript{v}3\textsuperscript{Founder,w-}} embryos (Rainbow Transgenic Flies, Inc., US) for $\phi$C31-mediated site-specific integration into the \emph{attP} landing site in the DmCa\textsubscript{v}3 locus.  The white-markers of the \emph{DmCa\textsubscript{v}3\textsuperscript{Gal4}} and \emph{DmCa\textsubscript{v}3\textsuperscript{Rescue}} lines were removed before \emph{DmCa\textsubscript{v}3\textsuperscript{Gal4}} and \emph{DmCa\textsubscript{v}3\textsuperscript{Rescue}} were backcrossed to \emph{w\textsuperscript{1118}} for more than 8 generations  After backcrossing, these lines were used for behavioral studies.  diff --git a/Methods_oocytes.tex b/Methods_oocytes.tex  index 09b4343..1af1a48 100644  --- a/Methods_oocytes.tex  +++ b/Methods_oocytes.tex 
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cRNA concentrations were estimated based on spectrophotometric optical density measurements at 260 nm.  Oocyte preparation from female \emph{Xenopus laevis} and injection of cRNAs was performed as previously reported\cite{kang:2006aa}.  GenBank accession numbers: rat Ca\textsubscript{v}3.1 ($\alpha$\textsubscript{1}G), AF027984\cite{PerezReyes:1998gn}; DmCa\textsubscript{v}3 C isoform, NP001096889.    diff --git a/Methods_qpcr.tex b/Methods_qpcr.tex  index 1e26f7d..514e5b3 100644  --- a/Methods_qpcr.tex  +++ b/Methods_qpcr.tex 
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Total RNA was extracted using Trizol (Invitrogen) and reverse transcribed using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific).  Quantitative real-time PCR was performed using the TOPreal qPCR 2x premix (RT500M, Enzynomics, South Korea).  Expression levels of \emph{period} were normalized to the level of \emph{rp49}.  Primers: \emph{per} (5'-GACCGAATCCCTGCTCAATA-3', 5'-GTGTCATTGGCGGACTTCTT-3'); (\seqsplit{5'-GACCGAATCCCTGCTCAATA-3'}, \seqsplit{5'-GTGTCATTGGCGGACTTCTT-3'});  \emph{rp49} (5'-ATGACCATCCGCCCAGCATA-3', 5'-GAGAACGCAGGCGACCGTTG-3'). (\seqsplit{5'-ATGACCATCCGCCCAGCATA-3'}, \seqsplit{5'-GAGAACGCAGGCGACCGTTG-3'}).  diff --git a/Results_clock.tex b/Results_clock.tex  index 7647c5e..97c11df 100644  --- a/Results_clock.tex  +++ b/Results_clock.tex 
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The reductions in rhythmicity we observed may be due to an increase in sleep during the transition between subjective day and subjective night.  Consistent with a functional circadian clock, transcriptional oscillation of \emph{period}, one of the core clock genes, is normal in \emph{DmCa\textsubscript{v}3\textsuperscript{Gal4}} flies (Fig. \ref{fig:4}b).  Thus, these results suggest that the increase in sleep caused by loss of DmCa\textsubscript{v}3 cannot be attributed to a direct effect on the molecular clock.    diff --git a/Results_expression.tex b/Results_expression.tex  index fa5e1ce..a10576f 100644  --- a/Results_expression.tex  +++ b/Results_expression.tex 
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After several failed attempts to generate an antibody that works well for immunohistochemistry, we decided to tag the endogenous DmCa\textsubscript{v}3 with GFP and then visualize its expression pattern in the adult brain.  First, we generated a founder line, (\emph{DmCa\textsubscript{v}3\textsuperscript{Founder, \emph{w+}}}}), using end-out homologous recombination to facilitate the versatile generation of a variety of different alleles\cite{Huang:2009ei} (Fig. \ref{fig:2}a).  In \emph{DmCa\textsubscript{v}3\textsuperscript{Founder, \emph{w+}}}} flies, an \emph{attP} landing site for $\phi$C31-mediated DNA integration and a floxed \emph{white\textsuperscript{+}} marker replace \sim2 $\sim$2  kb of genomic DNA surrounding the first coding exon of DmCa\textsubscript{v}3. Next, we removed the \emph{white\textsuperscript{+}} marker from the \emph{DmCa\textsubscript{v}3\textsuperscript{Founder, \emph{w+}}} line by Cre-mediated recombination to generate \emph{DmCa\textsubscript{v}3\textsuperscript{Founder, \emph{w-}}}.  We then used the $\phi$C31 integrase to insert into the \emph{attP} landing site of \emph{DmCa\textsubscript{v}3\textsuperscript{Founder, \emph{w-}}} an \emph{attB}vector (\emph{pGE-attB\textsuperscript{GMR}}) containing the deleted genomic region plus an additional GFP coding sequence and linker sequence in-frame before the start codon of DmCa\textsubscript{v}3.   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. 
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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 \emph{GFP::DmCa\textsubscript{v}3}, \emph{DmCa\textsubscript{v}3\textsuperscript{Gal4}} drives the expression of a membrane-tethered mCherry (\emph{UAS-mCD8-ChRFP}) broadly across the brain (Fig. \ref{fig:S1}a).  The \emph{DmCa\textsubscript{v}3\textsuperscript{Gal4}\textgreater{}mCherry} \emph{DmCa\textsubscript{v}3\textsuperscript{Gal4}$>${}mCherry}  and \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 ec2ee6a..e2acaf1 100644  --- a/Results_knockdown.tex  +++ b/Results_knockdown.tex 
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Finally, we sought to narrow down the sleep-regulating role of DmCa\textsubscript{v}3 to a specific brain region or circuit.  We used a range of neuronal Gal4 drivers that cover known sleep centers to knockdown DmCa\textsubscript{v}3, but none of them were capable of significantly altering sleep (Fig. \ref{fig:S5}).  This suggests DmCa\textsubscript{v}3 may function in novel sleep circuits.      diff --git a/Results_sleep.tex b/Results_sleep.tex  index d4852ac..791780d 100644  --- a/Results_sleep.tex  +++ b/Results_sleep.tex 
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To confirm that this elevated sleep phenotype is specific to DmCa\textsubscript{v}3 loss-of-function, we generated three independent deletion mutants via imprecise P-element excision.  As expected, all three deletion mutants as well as a trans-heterozygous mutants ($\Delta$3/$\Delta$115) show increased sleep, especially in constant darkness (Fig. \ref{fig:S2}).  In addition, knockdown of DmCa\textsubscript{v}3 in its own neurons (\emph{DmCa\textsubscript{v}3\textsuperscript{Gal4}\textgreater{}UAS-DmCa\textsubscript{v}3-IR}) (\emph{DmCa\textsubscript{v}3\textsuperscript{Gal4}$>${}UAS-DmCa\textsubscript{v}3-IR})  increases sleep after the third day of continuous darkness (Fig. \ref{fig:S3}). Together, these results implicate DmCa\textsubscript{v}3 as a novel sleep-inhibitor of fly sleep.    diff --git a/acknowledgements.tex b/acknowledgements.tex  index beb958f..759374b 100644  --- a/acknowledgements.tex  +++ b/acknowledgements.tex 
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\section*{Acknowledgements}  This work was supported bygrants to D.K. (20120008795 and 2012 K001117) from  the National Leading Priority  Research Laboratory Centers  Program and of the National Research Foundation of the Republic of Korea (2012-0006690) to J.L.. It was also supported by  the KAIST Future Systems Healthcare Project of the Ministry of Science, ICT and Future Planningand  to J.C. (NRF-2015-021435) D.K.  and W.D.J. (2013R1A1A2011339) grants  from the National Research Foundation(NRF)  of the Republic of Korea. Korea to D.K. (2014R1A2A1A12067558 and 2011-0028772), J.C. (NRF-2015-021435)and W.D.J. (2013R1A1A2011339).  diff --git a/discussion.tex b/discussion.tex  index fcb0c21..8ae9823 100644  --- a/discussion.tex  +++ b/discussion.tex 
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In this study, we cloned the only voltage-gated T-type Ca\textsuperscript{2+} channel from \emph{Drosophila}, DmCa\textsubscript{v}3.  DmCa\textsubscript{v}3 is the largest T-type channel cloned to date, measuring 3205 amino acids\cite{senatore:2010aa}.  Electrophysiological characterization of DmCa\textsubscript{v}3 in \emph{Xenopus} oocytes showed that DmCa\textsubscript{v}3 has the hallmark properties of a T-type channel: low-threshold activation at around \textminus60 $-$60  mV, a maximal current output at \textminus20 $-$20  mV, transient current kinetics elicited by a step-pulse protocol producing a ``criss-crossing'' pattern, and slow deactivation of tail currents (Fig. \ref{fig:1}). These biophysical properties are also consistent with previous studies that implicated DmCa\textsubscript{v}3 in low-voltage-activated (LVA) currents in both the central and peripheral nervous systems of the fly\cite{Ryglewski:2012jk, Iniguez:2013ib}.  Mammalian genomes contain three T-type Ca\textsuperscript{2+}channel genes (i.e., Ca\textsubscript{v}3.1-3.3), while the fly genome contains only one.  diff --git a/figures/Fig_2/caption.tex b/figures/Fig_2/caption.tex  index d51dbc7..a22708e 100644  --- a/figures/Fig_2/caption.tex  +++ b/figures/Fig_2/caption.tex 
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\textbf{(g)} Expression in the protocerebral bridge (PB) of the central complex.   \textbf{(h)} Expression in the mushroom body (MB) calyx.   Neuropils are counter-stained with the nc82 antibody ($\alpha$-Bruchpilot, magenta).    diff --git a/figures/Fig_3/caption.tex b/figures/Fig_3/caption.tex  index e83baf4..0ad0c07 100644  --- a/figures/Fig_3/caption.tex  +++ b/figures/Fig_3/caption.tex 
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Boxplot whiskers extend to the highest and lowest values that fall within 1.5 $\times$ IQR of the upper and lower quartiles.  All indications of statistical significance were determined using Welch's ANOVA followed by the Games-Howell post hoc test.  * \emph{p}$<$0.05, ** \emph{p}$<$0.01, *** \emph{p}$<$0.001.      diff --git a/figures/Fig_4/caption.tex b/figures/Fig_4/caption.tex  index 192627d..d2ede10 100644  --- a/figures/Fig_4/caption.tex  +++ b/figures/Fig_4/caption.tex 
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\textbf{(b)} Transcriptional oscillation of the \emph{period} gene in \emph{DmCa\textsubscript{v}3\textsuperscript{Gal4}} under DD conditions. Black and red lines denote \emph{w\textsuperscript{1118}} and \emph{DmCa\textsubscript{v}3\textsuperscript{Gal4}}, respectively.  \emph{rp49} was used for normalization.  a.u., arbitrary unit.  Data are presented as means $\pm$ standard error of the mean (SEM). diff --git a/figures/Fig_5/caption.tex b/figures/Fig_5/caption.tex  index 6b5425e..188eaea 100644  --- a/figures/Fig_5/caption.tex  +++ b/figures/Fig_5/caption.tex 
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\textbf{(b)} Quantification of average total sleep over two days of light-dark cycles (LD) and two days of continuous darkness (DD).  Data are presented as means $\pm$ standard error of the mean (SEM) and analyzed via one-way ANOVA followed by the Tukey-HSD post hoc test.  ***\emph{p}$<$0.001    diff --git a/figures/Fig_S1/caption.tex b/figures/Fig_S1/caption.tex  index 207de85..8ad3698 100644  --- a/figures/Fig_S1/caption.tex  +++ b/figures/Fig_S1/caption.tex 
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\textbf{(b-c)} Location corresponds to the boxed areas in (a).  \textbf{(b)} Expression in the ellipsoid body (EB), fan-shaped body (FB), and noduli (NO).  \textbf{(c)} Expression in the protocerebral bridge (PB), the mushroom body (MB) calyx, and the MB Kenyon cells.      diff --git a/figures/Fig_S3/caption.tex b/figures/Fig_S3/caption.tex  index 0ac64a1..ed43e79 100644  --- a/figures/Fig_S3/caption.tex  +++ b/figures/Fig_S3/caption.tex 
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Data are presented as means $\pm$ standard error of the mean (SEM).  Significance was determined with the one-way ANOVA followed by Tukey-HSD post hoc tests.  **\emph{p}$<$0.01, ***\emph{p}$<$0.001.    diff --git a/figures/Fig_S4/caption.tex b/figures/Fig_S4/caption.tex  index 7b8e32f..ba013ca 100644  --- a/figures/Fig_S4/caption.tex  +++ b/figures/Fig_S4/caption.tex 
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Data are presented as means $\pm$ standard error of the mean (SEM).  Significance was determined using the one-way ANOVA followed by Tukey-HSD post hoc tests.  *\emph{p}$<$0.05.    diff --git a/figures/Fig_S5/caption.tex b/figures/Fig_S5/caption.tex  index 2fd7289..c90215f 100644  --- a/figures/Fig_S5/caption.tex  +++ b/figures/Fig_S5/caption.tex 
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\\  \textbf {(a)} Average total sleep over two days of 12h:12h light-dark cycles (LD).  \textbf {(b)} Average total sleep over two days of continuous darkness (DD).  White, grey, and black bars denote \emph{UAS-DmCa\textsubscript{v}3-IR/+}, \emph{Gal4/+} and \emph{Gal4$>$DmCa\textsubscript{v}3-IR}, respectively (n=21-83). (n=21--83).  PI, pars intercerebralis, MB, mushroom body.   Data are presented as means $\pm$ standard error of the mean (SEM).  Statistical significance was determined using Welch's ANOVA followed by the Games-Howell post hoc test.