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\subsection*{DmCa\textsubscript{v}3 is broadly expressed in the adult brain}  \textbf{Expression pattern of GFP::DmCa\textsubscript{v}3 in adult brain}\\  To determine the expression pattern of DmCa\textsubscript{v}3 in the adult brain, wefirst  generated a \emph{white\textsuperscript{+}} DmCa\textsubscript{v}3\textsuperscript{Founder} allele in which a $\phi$C31 attP landing site and a floxed \emph{mini-white} marker replaces the first coding exon of GFP-tagged  DmCa\textsubscript{v}3via homologous recombination\cite{Huang:2009ei}.  We then removed the \emph{mini-white} marker via Cre-mediated recombination to produce a \emph{white\textsuperscript{-}} DmCa\textsubscript{v}3\textsuperscript{Founder}  allele. Finally, using $\phi$C31-mediated integration, First,  we generated a GFP-tagged DmCa\textsubscript{v}3 knock-in allele, GFP::DmCa\textsubscript{v}3, founder line (\emph{DmCa\textsubscript{v}3\textsuperscript{Founder}})  by inserting the end-out homologous recombination for versatile allele generation\cite{Huang:2009ei} (Fig. \cite{fig:2}a).  In \emph{DmCa\textsubscript{v}3\textsuperscript{Founder}}, about 2 kb of genomic region including first  coding sequence exon was replaced by \emph{attP} site  for $\phi$C31-mediated DNA integration and floxed \emph{white\textsuperscript{+}} marker.   After removing \emph{white\textsuperscript{+}} marker in the \emph{DmCa\textsubscript{v}3\textsuperscript{Founder}} by Cre-mediated recombination, \emph{attB} vector (\emph{pGE-attB\textsuperscript{GMR}}) containing deleted genomic region with  GFP in-frame immediately after and linker sequences inserted before  thetranscriptional  start codon in frame was integrated into \emph{attP}  site of the first founder line by $\phi$C31 integrase.   Integration resulted in  DmCa\textsubscript{v}3 exon (Fig. \ref{fig:S1}). tagged with GFP at its N-terminal end (\emph{GFP::DmCa\textsubscript{v}3}).  In GFP::DmCa\textsubscript{v}3 \emph{GFP::DmCa\textsubscript{v}3}  flies, GFP signal appears broadly across the brain, with the brain (Fig. \cite{fig:2}b).  Expressed region includes well-structured neuropils like  antennal lobes, lobe,  mushroom body calyces, body, central complex in central brain (Fig. \cite{fig:2}c-h)  andthe  optic lobes all showing strong staining (Fig. \ref{fig:2}). lobe as well as less-structured neuropil regions.  The strongest expression, though, is expression was detected  in the central complex, which comprises the ellipsoid body, complex consisting four substructures of  fan-shaped body, noduli, ellipsoid body, noduli  and protocerebral bridge (Fig. ).  GFP::DmCa\textsubscript{v}3 channels are also expressed broadly 2e and g) and the expression was predominent  in less structured neuropils including subesophageal zone (SEZ), superior neuropil (SNP), inferior neuropil (INP), ventrolateral neuropil (VLNP) ventral part of fan-shaped body  and ventromedial neuropil (VMNP). ventral noduli (Fig. \cite{fig:2}e).   In mushroom body, \emph{GFP::DmCa\textsubscript{v}3} was localized in postsynapse-rich calyx in dorso-posterior brain (Fig. 2h) rather than axonal lobes in anterior brain (Fig. \cite{fig:2}d).  We observed \emph{GFP::DmCa\textsubscript{v}3} expression only in the posterior part of peduncle, a fiber track of mushroom body joining the posterior calyx and anterior lobes (Fig. \cite{fig:2}f).  These results suggest strict regulation of 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, DmCa\textsubscript{v}3\textsuperscript{Gal4}.  In DmCa\textsubscript{v}3\textsuperscript{Gal4}, the Gal4 coding sequence replaces the first coding exon of DmCa\textsubscript{v}3 and its flanking introns to drive GAL4 expression under the control of the endogenous DmCa\textsubscript{v}3 promoter (Fig. \ref{fig:S1}). 
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