changes in environmental conditions can have a significant impact on the development and function of the brain .
neural stem cells ( nscs ) integrate both local and systemic signals to modulate their rate and extent of proliferation to meet the needs of the organism ( kokovay et  al . , 2008 ) .
these quiescent nscs are reactivated in response to a variety of metabolic stimuli ( rafalski and brunet , 2011 ) .
understanding how systemic and metabolic signals are sensed by the brain and converted into specific neural stem cell behaviors is essential to deciphering how the brain adapts to a changing environment . in drosophila , nscs enter quiescence at the end of embryogenesis and are reactivated during early larval life in response to feeding ( britton and edgar , 1998 ; truman and bate , 1988 ; figure  1a ) .
amino acid availability is sensed by the fat body , the functional equivalent of the mammalian liver and adipose tissue ( andersen et  al . , 2013 ; colombani et  al . , 2003 ) .
the fat body sends an as - yet - unidentified signal , or signals , to the brain to induce the production and secretion of insulin - like peptides ( dilps ) by blood - brain barrier ( bbb ) glial cells .
dilps act locally to trigger the insulin / insulin - like growth factor receptor pathway in underlying nscs ( chell and brand , 2010 ; sousa - nunes et  al . , 2011 ) .
nsc reactivation occurs synchronously in all neurogenic zones of the cns , suggesting that bbb glial cells and/or nscs are linked by an intercellular signaling mechanism .
gap junctions are intercellular channels formed by the juxtaposition of connexin hexamers ( segretain and falk , 2004 ) .
gap junctions are found throughout the mammalian brain and are important regulators of stem cell behavior , controlling self - renewal , survival , and aging ( kar et  al . , 2012 ; wong et  al . ,
we show that gap junction proteins play a key role in the nutrient - dependent reactivation of dormant neural stem cells in the drosophila brain .
interestingly , gap junction proteins are required in the bbb glia , but not in neural stem cells , for reactivation .
we show that gap junction proteins coordinate nutrient - dependent calcium oscillations within the bbb and are required for the production and secretion of insulin - like peptides .
gap junction proteins thus enable the synchronous reactivation of quiescent stem cells throughout the cns .
to assess whether gap junctions play a role in nsc reactivation , we systematically targeted each of the eight members of the innexin ( inx ) family ( bauer et  al . , 2005 ) , the drosophila functional equivalents of connexins and pannexins ( phelan , 2005 ; figure  s1a available online ) , by rnai in either nscs or glia ( table s1 ) .
however , knockdown of innexin 1 ( inx1 ) or innexin 2 ( inx2 ) in glia gave a striking phenotype in which brain size is dramatically reduced ( figures 1b1d ) without affecting overall body size ( data not shown ) .
this suggests that the inx phenotype is not the result of a systemic growth defect but that inx1 and inx2 have a specific role in the brain .
we checked the specificy of inx1 and inx2 using in  silico methods ( naito et  al . , 2005 ) , which predict no off - targets .
, 2013 , who showed that targeted rnai against inx1 ( ogre ) or inx2 in glia disrupts development of the larval nervous system and leads to adult behavioral phenotypes .
we demonstrate that innexins are not required to link nscs either to each other or to glial cells . instead
, inx1 and inx2 are required within the glial population alone for brain development . to understand how glial gap junctions regulate growth in the cns , we first examined nsc behavior after inx1 or inx2 knockdown at different time points during the process of nsc reactivation .
knockdown of inx1 or inx2 in glia did not affect the number of nscs in the ventral nerve cord ( vnc ; figure  1e ) , demonstrating that the phenotype is not due to the loss of nscs prior to nsc reactivation ( 0  hr after larval hatching , alh0 ) .
next we assessed cell diameter because one of the earliest events in nsc exit from quiescence is cell enlargement ( chell and brand , 2010 ; sousa - nunes et  al . ,
we found that nsc diameter is markedly reduced ( alh24 ) after inx1 or inx2 knockdown in glia ( figure  1f ) .
we assayed the mitotic marker phosphohistone h3 ( ph3 ) before nsc reactivation ( alh0 ) , just after reactivation ( alh48 ) and at a time when wild - type nscs are cycling actively ( alh72 ) .
knockdown of either inx1 or inx2 in glial cells resulted in a severe reduction in the number of dividing nscs at all times ( figure  1 g ) .
we found that nsc enlargement and entry into mitosis were also dramatically impaired in inx1 and inx2 mutants ( inx1 and inx2 , respectively , see figures s1b and s1c ) , and that reactivation could be rescued in inx2 mutants by glial expression of inx2 ( figures s1d  s1e ) .
we conclude that inx1 and inx2 are required in the glia for nsc exit from quiescence .
gap junction proteins ( connexins , pannexins , or innexins ) are classically involved in forming intercellular channels or hemi - channels , which enable exchange between the cytoplasm and the extracellular medium .
evidence also exists for channel - independent roles , such as cell adhesion and direct gene regulation ( reviewed in dbouk et  al . , 2009 ; elias and kriegstein , 2008 ) .
to test if channel function is important for nsc reactivation , we treated brains in culture with carbenoxolone , a classic blocker of gap junction channels and hemi - channels ( giaume and theis , 2010 ; see supplemental experimental procedures ) .
carbenoxolone completely blocked nsc reactivation ( figures 2a2c ) , implying a channel role for inx1 and/or inx2 , the only innexins required for nsc reactivation .
we also found that protein fusions that interfere with the folding of the innexin n - terminal domain ( gfp - inx1 and rfp - inx2 ) , which is essential for channel formation ( nakagawa et  al . , 2010 ) , act as dominant - negative mutants ( figures s2a  s2c ; data not shown for inx2 ) .
this suggests that the function of inx1 and inx2 in glia is channel - based .
inx1 and inx2 could be part of the same channel or form two distinct channels , performing different functions that are both required for nsc reactivation .
gap junction channels are formed by the apposition of connexons ( innexons in drosophila ) on adjacent cells .
connexons can be homomeric , formed from six molecules of a single subtype of connexin , or heteromeric , formed from different subtypes . in the larval vnc
prior to nsc reactivation ( alh7 ) , inx1 and inx2 were strongly expressed in glia and colocalized in plaques typical of gap junctions ( segretain and falk , 2004 ; figure  2d ) .
super - resolution microscopy ( dobbie et  al . , 2011 ) further demonstrated the tight association of inx1 and inx2 , using tagged fusion proteins ( figures 2e2e ) .
this close association is present from hatching and is not lost under starvation conditions ( figures s2d and s2e ) , demonstrating that formation of the complex is not driven by nutrition .
we found that most inx1 staining was lost after knockdown of inx2 in glia , and vice versa .
this suggests that inx1 and inx2 localization is interdependent ( figures s2f  s2h ) and that inx1 and inx2 form heteromeric innexons ( segretain and falk , 2004 ) rather than independent gap junction channels ( lehmann et  al . , 2006 ) .
inx1 and inx2 have been shown to form functional heteromeric channels in paired xenopus oocytes ( holcroft et  al . , 2013 ) .
we conclude , therefore , that inx1 and inx2 form heteromeric channels or hemi - channels in the glia .
by alh24 , when reactivation has taken place , inx1 and inx2 are still expressed but they no longer colocalize ( figure  2f ) , suggesting that formation of inx1/inx2 channels is  temporally regulated .
consistent with this observation , we discovered that the temporal requirement for inx1 and inx2 function in nsc reactivation is between alh0 and alh24 ( figure  2 g , table s2 , and data not shown ) .
therefore , the formation and maintenance of inx1 and inx2 heteromeric channels are developmentally regulated and coincide with the time when the innexins are required for nsc reactivation .
inx1/inx2 channels are required in glia to transmit nutritional stimuli to quiescent nscs , they are likely to be found , therefore , in cells situated between the nscs and the exterior of the brain . to determine in which glial cells inx1/inx2 are required , we knocked down inx1/inx2 in different glial populations using subtype - restricted gal4 drivers to drive rnai or express dominant - negative constructs ( table s3 ) .
we found that inx function is necessary within the subperineurial glia because knockdown in this glial subtype alone phenocopies knockdown in the entire glial population ( table s3 and figures s3a and s3b ) , preventing nsc reactivation ( figures 3a3d ) .
the subperineurial glia and the perineurial glia constitute the drosophila bbb ( stork et  al . , 2008 ) .
in vertebrates , the bbb consists of a single layer of vascular endothelium closely associated with astrocytic glia ( daneman , 2012 ) .
the bbb shields the brain from the external environment owing to tight junctions between endothelial cells .
it acts as a selective sieve to reject potentially neurotoxic factors but allow the passage of nutrients , ions , or other signals to maintain brain homeostasis ( daneman , 2012 ) .
the drosophila bbb exhibits similar neuroprotective strategies to its vertebrate counterpart , including a layer that limits the diffusion of neurotoxic factors , and an array of conserved transporters that regulates bbb permeability ( daneman and barres , 2005 ; desalvo et  al . , 2011 ; mayer et  al . ,
the subperineurial glia are large , flat polyploid ( unhavaithaya and orr - weaver , 2012 ) cells ( figures 3e3e ) that envelop the brain ( figure  3f ) and are closely apposed to the nscs ( figures 3f and 3f ) .
they isolate the brain from the hemolymph ( the drosophila equivalent of blood ) by virtue of lateral septate junctions ( figures 3e and 3e ; schwabe et  al . , 2005 ; stork et  al . ,
knockdown of inx did not disrupt the septate junctions ( figures s3c and s3d ) , and we were not able to see a change in dextran dye penetration ( data not shown ; pinsonneault et  al . , 2011 ; schwabe et  al . ,
although weak permeability defects are difficult to detect at this stage and can not be excluded , they do not prevent nsc reactivation ( moody mutant ( bainton et  al . , 2005 ) , data not shown ) .
these data suggest that the inx mutant phenotype is not due to an impaired , leaky bbb . using super - resolution microscopy , we detected inx1 ( and inx2 , data not shown ) along the bbb membranes and the septate junctions lining the lateral cell membranes ( figures 3 g and s3e ) .
we conclude that inx1/inx2 channels are required autonomously in the bbb glial cells for nsc reactivation ( figure  3h ) .
nsc reactivation requires the expression and secretion of insulin - like peptides , dilps , by bbb glial cells ( chell and brand , 2010 ) .
of the eight identified insulin - like peptides in drosophila , dilp6 transcription was shown to increase dramatically in the cns upon feeding . furthermore ,
when larvae were starved , forced expression of dilp6 in the glia was able to rescue nsc reactivation ( chell and brand , 2010 ) .
dilp6 binds to the insulin receptor ( inr ) on nscs , activating the pi3k / akt pathway and inducing exit from quiescence ( chell and brand , 2010 ) .
we found a significant decrease in dilp6 transcription after knocking down both inx1 and inx2 in the  glia ( figure  s4a ) .
, we expressed a tagged , functional version of dilp6 ( dilp6-flag ) in the bbb glia .
we found that dilp6 secretion from the bbb glia was strongly impaired in inx1 loss - of - function mutants ( figures 4a , 4b , and 4d ) .
secretion of dilp6 was similarly impaired upon starvation ( figures 4a , 4c , and 4e ) .
therefore , both the expression and secretion of dilp6 are regulated by nutrition and depend on gap junction proteins . nsc reactivation in inx mutants was rescued by forced expression of dilp6 in glia ( figures 4f4h , see arrowheads ) , as shown by the recovery of brain volume ( 80% of the brains , figure  4i ) and of nsc diameter ( figure  4j ) .
direct activation of the pi3k / akt pathway in nscs also resulted in rescue of brain volume and nsc enlargement and entry into mitosis ( figures s4b  s4c ) .
we conclude that gap junction proteins in the bbb glia are required to activate insulin signaling and induce nsc reactivation .
secretion of insulin by the pancreas is induced by glucose , leading to synchronized calcium oscillations within gap junction - coupled beta cells and insulin exocytosis ( macdonald and rorsman , 2006 ) .
gap junctions enable the passage of secondary messengers that either trigger the release of calcium from intracellular stores or the influx of calcium from the extracellular environment ( laude and simpson , 2009 ; leybaert and sanderson , 2012 ; orellana et  al .
inhibits coordinated intercellular calcium signaling ( leybaert and sanderson , 2012 ; orellana et  al . , 2012 ) .
gap junction proteins are thus an important means of transmitting calcium waves . to investigate whether calcium signaling plays a role in gap junction - mediated nsc reactivation , we expressed a calcium sensor , gcamp3 ( tian et  al . , 2009 ) , in the bbb glia ( figure  5a ; figures s5a
the bbb glia of feeding larvae exhibited clear calcium oscillations ( movie s1 ; figure  5b ) .
the bbb glia pulsed simultaneously , suggesting that calcium oscillations are coordinated across the entire cns .
individual cell tracking showed that glial calcium oscillations exhibited striking synchrony ( figures 5b and 5b ) in all brains analyzed ( n  = 15 , six are displayed ; figure  s5d ) . to further assess the extent of calcium oscillation coordination within the bbb glia under these conditions , we performed correlation analysis ( see experimental procedures ) for 20 regions of interest ( roi ) chosen at random within the bbb glial layer ( figures 5c , s5e , and s5e ) . in fed larvae before reactivation ,
the central correlation peak ( coefficient 1 ) demonstrates the synchronicity of calcium oscillations within the bbb layer .
additional peaks on each side reveal that this synchronicity repeats ( see experimental procedures for details ) .
none of the mutant brains ( n  = 11 ) showed coordinated calcium oscillations ( movie s2 ; figure  5d ; figure  s5f ) .
instead , bbb glial cells pulse independently , with no coordination between neighboring cells ( figures 5d and 5d ) .
we conclude that inx1/inx2 gap junctions are required to coordinate synchronous calcium oscillations within the bbb glia . in accordance with our observation ,
the graph of correlation coefficient for inx1 mutants established the total absence of synchronicity between bbb glial cells ( figures 5e , s5 g , and s5g ) , showing that gap junctions are required for propagating calcium oscillations within the bbb . to assess whether calcium oscillations in the bbb are induced by a nutritional stimulus , we first assayed calcium dynamics in the bbb glia of newly hatched larvae ( alh0 ) , before they started to feed .
the calcium oscillations differed both in extent and frequency from those seen in fed larvae ( n  = 9 ; movie s3 ; figures 6a6a ; figure  s6a ) .
correlation analysis revealed a partial coordination within the bbb glia ( central correlation peak with a coefficient of 0.5 , figures 6b , s6b , and s6b ) , strengthening the idea that nutrition is important for extending and establishing robust calcium synchronicity in the bbb .
next , we assessed calcium dynamics after starvation , specifically the absence of essential amino acids .
calcium dynamics in the bbb of starved larvae resembled inx1 mutant brains ( movie s4 ; figure  6c ) .
synchronous calcium oscillations were completely abolished in all brains examined ( n  = 19 , figure  s6c ) .
individual bbb cells displayed some calcium pulses ( figures 6c and 6c ) , but with different profiles to those seen in fed larvae .
in addition , correlation analysis of starved larvae showed very weak synchronicity ( figures 6d , s6d , and s6d ) .
this suggests that the synchronous , nutrition - dependent , calcium oscillations are lost upon starvation .
importantly , neither inx1 nor inx2 is lost under starvation conditions ( figure  s2e ) .
we conclude that nutrition , in particular essential amino acids , shape calcium dynamics . upon feeding ,
we assayed whether glial calcium oscillations arise from the release of intracellular calcium , or from the influx of extracellular calcium , and how these influence nsc reactivation .
first , we assessed the importance of the inositol - triphosphate ( ip3 ) pathway .
stimulation of g - coupled receptors by a wide range of signals activates phospholipase c , leading to the production of ip3 from cleaved pip2 .
ip3 then binds to its receptor ( ins3pr ) , a ligand - gated ca channel found on the surface of the er , releasing intracellular calcium .
we knocked down ins3pr ( itpr in drosophila ) in the bbb glia by rnai ( chorna and hasan , 2012 ) . both nsc enlargement and proliferation were strongly impaired ( figures 7a7d ) .
membrane depolarization triggers the entry of extracellular calcium via voltage - gated calcium channels ( catterall , 2011 ; leybaert and sanderson , 2012 ) , whereas hyperpolarization prevents it .
we hyperpolarized bbb membranes by expressing the inward - rectifying potassium channel , kir2.1 ( baines et  al . ,
bbb hyperpolarization blocks nsc reactivation dramatically , as revealed by the complete failure of both enlargement and mitotic re - entry ( figures 7e7h ) .
interestingly , the mushroom body neuroblasts , a small group of central brain nscs that do not undergo quiescence and reactivation , are not affected by bbb hyperpolarization , suggesting that nutrition - dependent nsc reactivation is specifically affected ( figures s7a and s7b , see arrowheads ) .
bbb hyperpolarization both decreases dilp6 mrna levels ( figure  s7c ) and dilp6 secretion ( figures s7d and s7e ) , similar to what is seen during starvation or gap junction loss - of - function ( see figure  s4a and figures 4a4e ) . in support of the role of calcium oscillations in reactivating nscs
, we found that overexpressing the calcium - binding protein , calmodulin , prevents nsc  reactivation ( figures s7c  s7f ) .
the nutrient - dependent reactivation of nscs in the drosophila brain demonstrates how nscs can adapt to environmental changes to fulfil the needs of the organism . here
, we show that gap junction proteins within the bbb glia are required for insulin expression and secretion , a prerequisite for nsc exit from quiescence ( figure  7i ) .
we demonstrate that gap junction proteins coordinate glial calcium oscillations that are required for nsc reactivation .
membrane depolarization is known to regulate exocytosis via calcium signaling ( fridlyand and philipson , 2011 ; stojilkovic , 2012 ; sdhof , 2012 ) , which controls stimulus - secretion coupling in secretory cells , such as in endocrine cells ( dolenek et  al . , 2011 ; gminard et  al . , 2009 ; stojilkovic , 2012 ; sdhof , 2012 ) .
we show that conditions that block calcium oscillation in the bbb glia ( the loss of gap junction proteins , starvation ) also impair insulin secretion .
the sequence of events leading to glial secretion of insulin bears a striking resemblance to the diet - induced release of insulin by the beta cells of the pancreas ( macdonald and rorsman , 2006 ) . in the pancreas , a nutritional stimulus
is sensed by gap junction - coupled beta cells , inducing depolarization resulting in synchronized calcium oscillation and insulin secretion .
loss of gap junction coupling results in uncoordinated calcium pulses . in drosophila inx loss of function mutants , individual subperineurial bbb
compared to starvation , in which the nutritional signal is absent and nsc reactivation can not occur , the scattered signals from individual bbb cells are able to induce delayed , asynchronous , reactivation in a small number of nscs ( see ph3 positive nscs in figures 1c1d ) .
we propose that gap junction function within the bbb enables glial insulin release to reach a threshold high enough to trigger nsc reactivation throughout the central nervous system . in both beta cells and bbb cells ,
however , sustained depolarization of  cells can lead to desensitization and a decline in insulin release ( willenborg et  al . ,
interestingly , we find that forced depolarization of bbb glia only mildly enhances nsc reactivation ( data not shown )
. this could be due to desensitization or it may be that the system is already maximally active .
insulin mrna levels are decreased after gap junction knockdown , both in pancreatic islets ( bosco et  al .
it remains to be determined if calcium oscillations can directly affect gene expression , as has been shown in other systems ( alonso and garca - sancho , 2011 ) .
insulin produced by the pancreas is distributed via the circulatory system , whereas glial insulin is secreted locally , directly to  underlying nscs .
glial insulin signaling is thus contained within the brain , enabling local , differential regulation of this organ .
the bbb acts both as a niche and as a protective barrier , providing specific factors directly to the stem cell while shielding  the brain from unwelcome systemic regulation . in the context  of  nsc reactivation , these two roles are conveniently complementary . in the vertebrate bbb , similar functions may be split between endothelial cells and astrocytic glia .
the vascular endothelium provides the barrier function , while astrocytic glia have a regulatory role in sensing and adjusting barrier permeability to various stimuli ( daneman , 2012 ) .
bbb endothelial cells can secrete cytokines , chemokines , and prostaglandins , suggesting that the bbb behaves like an endocrine tissue ( banks , 2012 ) .
interestingly , calcium oscillations have been observed in cultured vertebrate bbb endothelial cells , but their function is largely unknown ( de bock et  al . , 2013 ) .
gap junction communication can influence stem cell behavior by directly coupling stem cells to each other or to supporting cells , such as found in a stem cell niche . in the brain ,
connexon - mediated communication has been reported to occur between progenitor cells , within astrocytic networks , and between radial glia and neurons or progenitor cells and astrocytes ( giaume et  al .
, 2010 ; lacar et  al . , 2011 ; nakase and naus , 2004 ; lo turco and kriegstein , 1991 ) .
the proliferation of neural progenitors and the formation of cortical layers in the mouse brain depend on an intercellular gap junction network ( malmersj et  al . , 2013 ) , and grafted human nscs integrate into organotypic cultures through connexin coupling ( jderstad et  al .
, we show that gap junction function within a niche , the bbb , can also influence nsc behavior .
the drosophila bbb is a protective and selective barrier as well as a signaling center that orchestrates major developmental and physiological events .
here we show that gap junction communication enables cells within the bbb to act as a concerted unit , leading to coordinated calcium signaling and insulin release .
similarities between the bbb in vertebrates and invertebrates suggest that our findings are likely to have broader significance .
the rnai and gal4 drivers used in this study are listed in tables s1 and s3 .
inx1 and moody - gal4 were kind gifts of p. phelan and r. bainton , respectively .
all glial knockdowns were performed with repo - gal4 as driver , unless stated otherwise .
the bbb driver was either moody - gal4 or mdr65-gal4 ( see for each experiment ) .
center values are averages . student s t test was performed using a threshold of p  < 0.05 ( confidence interval of 95% ) , represented by .
all tests performed in this study were two - sided and with samples of same variance , unless stated otherwise .
for rescue of inx1 mutant by glial expression of dilp6 , we used a one - way anova test , with p  < 0.05 represented by .
whisker plots were drawn using the minimum , quartile 1 , median , quartile 3 , and maximum of each condition s sample .
in addition , a student s t test based on a sample s average and sd was used to generate p values with the same criteria as above .
we used tub - gal80 , inx1 
 ; repo - gal4 to achieve conditional expression of inx1 . at 25c
( permissive temperature for gal80 ) , larvae showed a wild - type phenotype , but at 30c ( restrictive temperature for gal80 ) , all larvae showed a mutant phenotype : lack of nsc reactivation and clear reduction in brain volume . in further experiments , we scored mutant phenotypes by brain volume only in third instar larvae .
twelve larvae were scored for each condition . in single temperature - shift experiments , 1  hr embryos or 1  hr first instar larvae
we coexpressed a tagged version of dilp6 ( dilp6-flag ) and a membrane marker ( mcd8-rfp ) in the bbb glia alone , in controls ( genotype moody - gal4 , uas - mcd8-rfp , uas - dilp6-flag ) and inx1 mutants ( genotype inx1/y ; moody - gal4 , uas - mcd8-rfp , uas - dilp6-flag ) .
all brains were imaged using the same confocal settings for dilp6-flag . for each vnc , the total dilp6 intensity ( t )
retained dilp6 intensity ( r ) was then measured as the dilp6 signal ( same threshold as previously ) colocalizing with  the bbb membrane ( rfp signal ) .
the threshold for the rfp signal was determined manually for each brain , to ensure selection of the whole membrane . due to the flatness of the bbb glia , the membrane signal represents most of  the cells , although some cytoplasmic signal could be missed .
the percentage of secreted dilp6 was calculated as percent secreted dilp6  = ( 1   r / t )  100 .
moody - gal4 , uas - kir2.1-gfp ; tubulin - gal80 flies were grown at permissive temperature until hatching , then switched to 30c from alh0 to alh24 .
two different conditions were analyzed : control ( genotype fm7 , dfd gmr yfp / y ; moody - gal4 , uas - mcd8-rfp , uas - gcamp3 ) and inx1 mutant ( genotype inx1/y ; moody - gal4 , uas - mcd8-rfp , uas - gcamp3 ) .
larvae were raised and staged at 29c , then fed or starved as described in the supplemental experimental procedures .
larvae were placed in a drop of voltalef oil on a 22  mm diameter welco dish , ventral side down .
a 13  mm diameter round coverslip was placed tilted on top and lowered until the larva was immobilized .
mounted larvae were imaged with a 40 oil immersion objective on an olympus inverted fv1000 .
gcamp and mcd8-rfp were imaged simultaneously because we did not detect any bleed - through . to measure the mean intensity of the whole plane over time ,
the mean intensity was measured for each point both for gcamp3 ( channel 1 , c1 ) and mcd8-rfp signals ( channel 2 , c2 ) .
we normalized changes in gcamp3 intensity against changes in mcd8-rfp intensities ( accounting mainly for loss of focal plane due to larval movement ) , and then normalized the ratio by its minimum over the whole movie , so that the baseline for all movies was always 1 .
the normalized gcamp3 signal is then : i(gcamp ) norm  = ( c1/c2 ) / ( c1/c2)min . this normalized ratio was plotted over time using spreadsheet software . to follow the behavior of calcium in different regions of the brain to check for oscillation coordination ,
rois were selected manually in the first frame of the movie , and their central coordinates recorded .
a 64   64 pixel tracking window was centered on each roi , and a 32   32 pixel subwindow in the middle of each tracking window was used for measuring intensity values .
the tracking operations were performed on the relatively stable red channel ( cell membrane ) rather than the more dynamic green channel ( the calcium signal to be measured ) .
the script moves the tracking window by calculating the correlation between the window region from one frame to the next .
the location of the peak of the correlation function indicates how far and in which direction to move the tracking window to keep it centered on the roi . to reduce tracking drift due to rounding errors ,
times - two upsampling was used . to further improve tracking accuracy , the image was low pass filtered to remove noise .
the upsampling and filtering were accomplished simultaneously by taking the product of the fast fourier transforms of the tracking window and extracting the low - frequency coefficients then zero padding before taking the inverse fast fourier transforms .
the intensity measurements were performed by summing the pixel values within the 32   32 pixel measuring window for both the red and green channels .
normalization was then applied as described above . the intensity signal for each roi ( computed and normalized as described above )
the baseline is calculated using a 50 sample running average filter and then subtracted out from the signal . the remaining signal
is then scaled so that the sum of the squares of the samples is one .
each remaining roi signal is correlated with the reference for shifts of 50 to  + 50 samples .
if a signal is identical to the reference , the correlation coefficient will peak at  + 1 for a shift of 0 .
if a signal is uncorrelated with the reference , then the correlation coefficient will take small random values .
 summaryneural stem cells in the adult brain exist primarily in a  quiescent state but are reactivated in response to  changing physiological conditions . how do stem cells sense and respond to metabolic changes ? in the drosophila cns , quiescent neural stem cells are reactivated synchronously in response to a nutritional stimulus . feeding triggers insulin production by blood - brain barrier glial cells , activating the insulin / insulin - like growth factor pathway in underlying neural stem cells and stimulating their growth and proliferation . here 
 we show that gap junctions in the blood - brain barrier glia mediate the influence of metabolic changes on stem cell behavior , enabling glia to respond to nutritional signals and reactivate quiescent stem cells . 
 we propose that gap junctions in the blood - brain barrier are required to translate metabolic signals into synchronized calcium pulses and insulin secretion . 
Introduction
Results
Discussion
Experimental Procedures