this requires that homeostatic processes maintain blood glucose concentration at a minimum of 5  mm to ensure sufficient provision of glucose to the brain ( unger and orci , 1981 ) . specialized glucose sensing cells present in peripheral locations , such as the hepatoportal vein area , and in the cns are activated by hypoglycemia and initiate a counterregulatory response to normalize glycemia ( chan and sherwin , 2013 , frizzell et  al . this response is mediated by activation of the autonomous nervous system and the hypothalamo - pituitary - adrenal axis , leading to the release in the bloodstream of glucagon secreted by pancreatic alpha cells , and of catecholamines and glucocorticoids secreted by the adrenal gland ( verberne et  al . , 2014 ) . the combined action of these hormones promotes hepatic glucose production , lipolysis in the adipose tissue , suppression of insulin secretion , and inhibition of glucose uptake in muscle and fat . these actions restore normoglycemia and glucose flux to the brain ( verberne et  al . , 2014 ) . the counterregulatory response to hypoglycemia is , thus , essential for survival . in type 1 and type 2 diabetic patients treated with insulin , the counterregulatory response to hypoglycemia is progressively blunted causing occasional hypoglycemia . antecedent hypoglycemic episodes then lead to a progressive loss of the hypoglycemia cognitive warning signals and to a reduced hormonal counterregulatory response . this condition , referred to as hypoglycemia associated autonomic failure ( haaf ) , augments the risks to develop hypoglycemic episodes of increased severity and is a major obstacle in the treatment of diabetes with insulin ( cryer , 2013 ) . hypoglycemia detection in the brain occurs within specialized brain areas , mainly the hypothalamus and the brainstem ( marty et  al . these brain structures contain neuron subpopulations activated by an increase or a fall in extracellular glucose concentrations and which are referred to as glucose excited ( ge ) or glucose inhibited ( gi ) neurons , respectively ( routh , 2002 ) . identification of these neurons is based on electrophysiological recording of their firing activity assessed in acute brain sections or using dispersed neuron populations . the molecular mechanisms of glucose sensing by ge or gi neurons are diverse ( thorens , 2012 ) . glucose sensing by ge neurons involves proteins also controlling glucose - induced insulin secretion by pancreatic beta - cells such as glut2 ( lamy et  al . , 2014 , glucokinase ( levin et  al . , 2008 , stanley et  al . , 2013 ) , and the katp channel ( evans et  al . , 2004 , miki et  al . the na / glucose co - transporters sglt1 and sglt3 may also contribute to glucose sensing by ge neurons ( omalley et  al . on the other hand , amp - activated protein kinase ( ampk ) plays a key role in activation of gi neurons by hypoglycemia ( alquier et  al . in contrast to ampk and the katp channel , which are present in all neurons , glut2 , glucokinase , and the sglts are present in specific , only partly overlapping neuronal subpopulations and may thus be used as markers of glucose - sensing neurons . identification of glucose sensing cells , in particular , those implicated in the adaptive response to hypoglycemia is of high importance to better understand the cause and possible management of haaf . so far , the mechanisms of central glucose sensing have been explored based on the targeted analysis of genes known to be involved in glucose metabolism or signaling . unbiased identification of genes controlling quantitative traits is , however , possible by screening genetic reference populations . the bxd recombinant inbred mice , which have been derived from the cross of dba/2j and c57bl6/j mice , form a particularly useful genetic reference population ( andreux et  al . , 2012 , chesler et  al . , 2004 , peirce et  al . , 2007 , rosen et  al . , 2007 ) . these mice have been successfully used to identify genes involved in various physiological processes such as sleep ( franken et  al . , 2001 ) , aging ( houtkooper et  al . , 2013 ) , blood pressure ( koutnikova et  al . , 2009 ) , or beta - cell function ( wong et  al . , 2013 ) . here , we aimed at identifying genes involved in the counterregulatory response by screening a bxd mouse panel for the glucagon response to 2-deoxy - d - glucose ( 2dg)-induced neuroglucopenia . this led to the identification of a significant qtl on  the distal part of chromosome 7 . to identify the causal gene in the qtl , we hypothesized that this gene should be expressed in the hypothalamus , and that its expression should  correlate with the glucagon trait . combining qtl analysis  and rna - sequencing ( seq ) data , we identified hypothalamic fgf15 as the  most likely candidate gene . the functional involvement of fgf15 was supported by showing that : ( 1 ) it is expressed in dorsomedial hypothalamus and perifornical neurons ; ( 2 ) intracerebroventricular ( i.c.v . ) delivery of fgf19 , the human ortholog of fgf15 , reduced 2dg - induced glucagon secretion through suppression of dorsal vagal complex neuron activation , and reduction of parasympathetic nervous activity ; and ( 3 ) silencing of fgf15 in the dmh increased 2dg - induced glucagon secretion . we searched for genomic intervals associated with glucagon secretion induced by 2dg - induced neuroglucopenia in a panel of 36 bxd mouse strains . groups of male mice were generated for each bxd strain and used when they reached 10  weeks of age . in a first experimental session , mice were injected intraperitoneally ( i.p . ) with a saline solution and blood was collected 30  min later for plasma preparation and storage . glucagon levels were measured by radioimmunoassay at the same time for all mice and the ratio between plasma glucagon after 2dg and nacl injections was calculated . this trait varies by 3-fold between bxd strains ( figure  1a ) with the lowest response ( 1.5-fold ) detected in bxd49 and the highest ( 4.7-fold ) in bxd50 . this trait was due to differences in stimulated plasma glucagon levels rather than to differences in basal glucagonemia ( figure  1b ) ; it was not correlated to differences in total pancreatic glucagon content across all the bxd strains ( figure  1c ) . this suggests that the variability in the secretion response results from differences in hypoglycemia detection and signaling to pancreatic alpha cells rather than from differences in pancreatic glucagon stores . quantitative trait analysis identified a whole genome significant qtl at the distal part of the chromosome 7 ( figure  1d ) . the likelihood ratio statistic ( lrs ) peak is centered on marker rs2304086585 ( lrs : 24.679 ; position : 151.6236 mb ) and flanked by markers c7.loc119 ( position : 147.5303 mb ) and rs31652431 ( position : 152.4411 mb ) ( figure  1e ) . this locus is 5 mb wide and it contains 128 genes ; it explains 49 percent of the variance of the glucagon trait . to narrow down the genes within the qtl , which could regulate glucagon secretion , we hypothesized , first , that these must be expressed in the hypothalamus where glucose sensitive neurons are located ( karnani and burdakov , 2011 , ritter et  al . , 2011 , routh , 2010 ) and , second , that their level of expression must correlate with the glucagon phenotype , assuming that they are non - coding polymorphisms . we thus performed transcriptomic analysis of the hypothalamus from all the bxd strains using mice that had not undergone qtl screening to avoid a possible impact of 2dg treatment on gene expression . rna was extracted from the hypothalamus of 36 mice for each strain and equal amounts of rna from each mouse were pooled for transcript profiling by rna sequencing analysis . we found that 65 of the 128 genes present in the chr7 locus were expressed in the hypothalamus . the hypothalamic expression level of 11 genes was significantly correlated to the glucagon trait ( table 1 ) . fgf15 mrna was the most significantly , and negatively , correlated to the glucagon phenotype ( r  = -0.571 ; p  = 2.73   10 ) ( table 1 ) . the second gene in this table , which shows a similar high correlation with the trait as fgf15 is gm17685 ( r  = 0562 ; p  = 3.58   10 ) . this encodes a long non - coding rna transcribed in reverse orientation from exon 2 of fgf15 , and its expression is highly correlated with that of fgf15 ( r  = 0.824 ; p  = 6.68   10 ) . the correlation with the glucagon trait of the other nine genes ( table 1 ) showed at least 10-fold less significant p values . furthermore , the fgf15 gene of the dba/2j and c57bl/6 mice encodes the same amino acid sequence , supporting differential expression of this gene as a possible underlying cause of the glucagon trait . we thus selected to further investigate fgf15 expression in the hypothalamus and its role in the response to neuroglucopenia . quantitative analysis of fgf15 expression in different brain nuclei showed highest expression in the hypothalamus ( average cycle threshold [ ct ] of 28.7 ) and lower expression in the hippocampus , thalamus , and brainstem , with almost undetectable levels in the cerebellum and cortex ( average cts of 29.6 , 30.1 , 30.1 , 34.2 , and 34.5 , respectively ) ( figure  2a ) . brain expression of fgf15 was , however , much lower than its expression in the ileum , its major site of expression ( average ct of 21.1 ) ( figure  2a ) ( inagaki et  al . , 2005 ) . expression of the fgf15 receptors fgfr1c , fgfr4 , and of the co - receptor klotho beta was found in all brain regions ( figures 2b2d ) . to determine the site of fgf15 expression , we performed in  situ hybridization analysis on hypothalamic sections . as shown in figures 2e2i , fgf15 expression was found mainly in the perifornical area ( pef ; figure  2e ) and the dorsomedial hypothalamus ( dmh ; figure  2 g ) . in situ hybridization using a control  probe derived from a bacterial gene , dapb , showed no staining ( figure  2h ) . no fgf15 transcripts were detected in the dorsal vagal complex of the brainstem ( figure  2i ) . detection of fgf15 in illeal villi is shown for comparison ( figure  2j ) . finally , double in  situ hybridization of fgf15 and of the neuronal marker neun showed that fgf15 was expressed in neurons ( figures 2k and 2l ) . to verify that fgf15 could regulate glucagon secretion through a central mechanism of action , we injected c57bl/6 mice i.c.v . with fgf19 ( the human ortholog of fgf15 , which shows high stability in  vivo and activates the same receptors ; owen et  al . , 2015 ) or an artificial cerebrospinal fluid ( acsf ) . injections of acsf or fgf19 ( figure  3a ; t  = 0 ) and was similarly increased 30  min after i.p . however , following 2dg - induced neuroglucopenia , the glucagon response was markedly lower in mice previously treated with i.c.v . next , we assessed hypothalamic fgf15 expression and 2dg - induced glucagon secretion in normal chow ( nc ) or high fat diet ( hfd ) fed mice , which show altered glucagon plasma levels ( burcelin et  al . , 2002 ) . mice fed a hfd for 4  weeks showed increased hypothalamic expression of fgf15 compared to nc fed mice ( figure  3f ) . when injected with 2dg , nc and hfd mice showed the same hyperglycemic response ( figure  3 g ) . the basal glucagonemia of both groups of mice were the same , but the plasma glucagon response to 2dg was markedly reduced in hfd fed mice ( figure  3h ) . thus , hfd feeding increased hypothalamic fgf15 expression and is correlated with reduced neuroglucopenia - induced glucagon secretion in agreement with the results of the genetic screen . to more directly determine whether the level of expression of fgf15 in the hypothalamus regulates glucagon secretion , we developed recombinant lentiviruses to express a control or an fgf15-specific short hairpin ( sh)rna . three shrnas were designed to target the fgf15 sequence and were subcloned in lentiviral vectors . these were first tested in hek293 t cells co - transfected with an fgf15-gfp expression plasmid . one vector induced efficient silencing of fgf15 mrna and protein expression ( figures 4a4c ) . this was used to generate a recombinant lentivirus , which effectively silenced fgf15 expression in transduction experiments ( figure  4d ) . this or a control lentivirus was then injected bilaterally in the dmh ( figure  4e ) . no difference in basal glycemia or in glycemic response to 2dg injection was observed between the two groups of mice ( figure  4f ) . however , neuroglucopenia - induced glucagon secretion was significantly increased ( 22% ) in mice with correct bilateral dmh injection of the silencing lentivirus as compared to mice injected with the control lentivirus ( figure  4 g ) or mice injected with fgf15-specific shrna lentivirus outside of the dmh ( figure  4 g ,  missed  ) . bilateral injection of the silencing lentivirus in the pef ( figure  4h ) did not impact glycemia before and 30  min after i.p . 2dg ( figure  4i ) , nor the neuroglucopenia - induced glucagon secretion ( figure  4h ) . taken together , these data show that fgf15 expression by dmh neurons has a negative impact on neuroglucopenia - induced glucagon secretion . glucagon secretion during hypoglycemia is triggered by the activation of both parasympathetic and sympathetic branches of the autonomic nervous system ( ans ) ( taborsky and mundinger , 2012 ) . fgf19 decreased the glucagon response to 2dg - induced neuroglucopenia , but had no effect in non - injected or i.p . saline injected mice , we measured sympathetic and parasympathetic nerve activities in response to i.p . parasympathetic nerve activity showed no differences between groups in the basal state ( figure  5a ) . after 2dg injection , its activity increased 2-fold in mice pre - treated with acsf , but remained unchanged in mice pre - treated with fgf19 ( figure  5a ) . sympathetic nerve activity showed no differences between the groups in the basal state or following 2dg injections ( figure  5b ) . the dorsal motor nucleus of the vagus ( dmnx ) is formed by the soma of the vagal neurons and is controlled by inputs from several brainstem and hypothalamic nuclei ( berthoud et  al . , 1990 , 2015 ) . to investigate whether fgf19 influences these neurons directly or indirectly , we quantified the number of perk1/2 ( activated by fgf19 binding to its receptor ) positive cells in the hypothalamus and the brainstem 30  min after i.c.v . we observed an increased number of perk1/2 positive cells in the arcuate nucleus ( arc ) after i.c.v . no increase in perk1/2 positive cells was observed in other hypothalamic nuclei , in the dorsal vagal complex ( dvc , which comprises the dmnx , the nucleus tractus solitarius [ nts ] , and the area postrema [ ap ] ) , nor in other brainstem areas . these results are consistent with a previous study ( marcelin et  al . , 2013 ) and suggest that the primary neurons involved in the effect of fgf19 are located within the arc . to determine whether fgf19 could control vagal neurons of  the dvc in an indirect manner , we injected mice i.c.v . with  acsf or fgf19 and 1  hr later i.p . with nacl or 2dg . thirty min later , the mice were killed and brain sections prepared for c - fos expression analysis by in  situ hybridization . 2dg induced a strong increase in c - fos expression in the nts and the dmnx . however , in mice , which had previously received fgf19 , 2dg - induced c - fos expression was markedly lower in both structures . these data further support a role for central fgf15 in modulating the sensitivity of the dvc to 2dg - induced neuroglucopenia and in reducing the stimulation of vagal activity . they also support a role of fgf15 neurons in controlling the dvc through an indirect mechanism that first controls arcuate neurons activity . the present unbiased approach identified hypothalamic fgf15 as a negative regulator of glucagon secretion in response to neuroglucopenia . in situ hybridization revealed that fgf15 is expressed by a subgroup of neurons of the dmh and pef . injection of fgf19 reduced , whereas shrna mediated silencing of fgf15 in the dmh increased neuroglucopenia - induced glucagon secretion . delivery of fgf19 prevented the increase in vagal activity induced by neuroglucopenia , and this was associated with a reduced activation of dvc neurons . as i.c.v . fgf19 activates perk1/2 only in hypothalamic arc neurons , our data suggest that hypothalamic fgf15 modulates dvc activity in an indirect manner . fgf15/19 belongs to the endocrine fibroblast growth factor family , which also includes fgf21 and fgf23 ( owen et  al . , 2015 ) . fgf15/19 expression in ileal cells is induced by the bile acids released in the intestinal lumen in the postprandial state through activation of the bile acid nuclear receptor fxr ( inagaki et  al . , 2005 ) . fgf15/19 binds to receptors consisting of fgfr1c or fgfr4 and the co - receptor klotho - beta ( owen et  al . , 2015 ) . in liver , ffg15/19 reduces bile acid synthesis through the inhibition of cyp7a1 ( jung et  al . , 2007 , it also induces glycogen synthesis through the activation of the ras - erk - p90rsk and inhibits gluconeogenesis via the creb - pgc1 pathway ( kir et  al . ffg15/19 has more recently been described to affect body weight , glucose tolerance , and insulin sensitivity through a central action ( marcelin et  al . , 2013 , , we found that fgf15 is also expressed in the hypothalamus through an unbiased search for genetic intervals associated with neuroglucopenia - induced glucagon secretion in recombinant inbred mice . among the 128 genes present in the identified qtl of chromosome 7 , rna - seq analysis revealed that 11 of these genes were expressed in the hypothalamus and expression of fgf15 , and , of the large intergenic noncoding rna ( linc)rna - encoding as lincrnas are implicated in the control of gene expression through a variety of mechanisms ( zhao and lin , 2015 ) , it could be hypothesized that gm17685 plays a regulatory role in fgf15 expression . testing this will , however , require future investigations . to localize fgf15 expression in the brain , we used a highly sensitive and specific in  situ hybridization technique . in the hypothalamus , expression of fgf15 was found in the pef and in the dmh , and co - in situ hybridization analysis with the neuronal marker neun revealed that it was expressed in neurons . no signal for fgf15 was identified in the dvc or in any other brainstem nuclei . to assess whether central fgf15 could impact neuroglucopenia - induced glucagon secretion , fgf19 decreased the glucagon response induced by 2dg , in agreement with the results of the genetic screen . we also observed in a model of high fat diet feeding an association between overexpression of hypothalamic fgf15 and a decrease in 2dg - induced glucagon secretion , lending further support to the negative correlation between hypothalamic fgf15 and glucagon secretion . however , the most convincing evidence for a direct link between hypothalamic fgf15 expression and glucagon secretion comes for the shrna - mediated silencing of fgf15 . this effect was observed when injection of the recombinant lentivirus was in the dmh , but not in the pef . because the pef is a rather large structure , it is possible that our injections were not sufficiently extensive over the entire pef structure to observe an effect . nevertheless , the critical observation is that injection of the recombinant lentivirus in the dmh was sufficient to increase 2dg - induced glucagon secretion , establishing a direct link between fgf15 in this structure and the control of glucagon secretion . fgf19 could improve glucose tolerance in hfd fed mice and ob / ob mice ( marcelin et  al . , 2013 , morton et  al . , 2013 ) . in ob / ob mice , the effect of fgf19 on glucose tolerance was associated with an increase in  insulin - independent glucose disposal and insulin sensitivity without changes in  insulin secretion . injection of fgf19 and was not modified by this treatment ( morton et  al . , 2013 ) . neuroglucopenia - induced glucagon secretion involves activation of the parasympathetic and sympathetic branches of the autonomic nervous system . during graded hypoglycemia development , the parasympathetic nerve is activated immediately when the glycemia falls under the 5  mm and sympathetic activity is increased at lower glycemic levels ( taborsky and mundinger , 2012 ) . here , we found that i.c.v . fgf19 dampens parasympathetic , but not sympathetic nerve activity during 2dg - induced neuroglucopenia , an observation that provides an explanation for the reduced glucagon secretion . moreover , the observation that 2dg - induced - glucagon secretion was not completely blunted after i.c.v . fgf19 can be explained by the fact that the sympathetic activity was not affected by i.c.v . this suggests that fgf19 specifically impairs the parasympathetic activity to reduce glucagon secretion during neuroglucopenia . the primary neurons activated by i.c.v . fgf19 and identified by immunofluorescence detection of perk1/2 are , however , present in the arc and not found in other hypothalamic or brainstem nuclei . this is in agreement with previous observations showing that fgf19 activates perk1/2 in the arc ( marcelin et  al . , 2013 ) . fgf19 blunted 2dg - induced neuronal activation in the nts and the dmnx as assessed by c - fos expression analysis . fgf19 can prevent activation of dvc neurons in response to neuroglucopenia , and our observations suggest that its action may be indirect through a first action on arc neurons . fgf19 induces perk1/2 in npy / agrp neurons , but fails to induce c - fos in pomc neurons ( marcelin et  al . thus , npy / agrp neurons could be the primary target neurons of fgf15 neurons of pef and/or dmh , and these neurons may then negatively control dvc neurons in a perk1/2-dependent manner . a role for the arc and npy / agrp neurons in mediating the effects of fgf15/19 can be supported by the fact that the melanocortin pathway has been shown to be necessary for the central effects of fgf19 on glucose tolerance ( marcelin et  al . whereas tracing approaches in mice expressing the cre recombinase under the control of fgf15 promoter should be used to confirm these observations , our data provide a first insight in a fgf15-dependent neural network , which involves an arc / dvc pathway to regulate glucagon secretion during neuroglucopenia . together , our genetic screen combined with gene expression analysis provides compelling evidence that hypothalamic fgf15 is negatively involved in the control by neuroglucopenia of glucagon secretion . fgf15 is expressed by discrete subpopulations of neurons in the pef and dmh and fgf15/19 blunts vagal firing activity and dampens the response of the dvc neurons to 2dg - induced neuroglucopenia , a response that involves activation of perk1/2 in arc neurons . collectively , these results , initiated by an unbiased search for novel genes involved in counterregulatory response to neuroglucopenia , reveal a new site of fgf15 expression and provide a mechanistic framework to elucidate fgf15 action in the control of parasympathetic nervous activity and glucagon secretion . they may provide new insight into the neuronal control of counterregulation and its deregulation in the etiology of haaf in type 1 and type 2 diabetic patients . bxd mouse strains were purchased from the center of phenogenomics of epfl ( lausanne ) . animals were housed on a 12  hr light / dark cycle and were fed a standard rodent chow diet ( diet 3436 , provimi kliba ) or a high - fat diet ( 235 hf , safe diets ) . number : 10 - 1247 - 10 , mercodia ) , and corticosterone ( cat . free plasma epinephrine was quantified as described ( dunand et  al . , 2013 ) . on the first experiment day , glycemia was taken at 9:00 a.m. and , if stable for 1  hr , the mice received at 10:00 am an i.p . total rna extraction from hypothalamus and brainstem samples was performed using the peqgold trifast method ( peqlab ) . rna were obtained from mice sacrificed during the morning in random fed conditions as for the qtl screening . rna integrity was verified with a 2100 bioanalyser ( agilent ) . for each bxd strain , equal amounts of rnas from 3 to 6 hypothalami were pooled in a final volume of 50  l . rna - seq libraries were prepared using 1  g of pooled rnas and the illumina truseq stranded mrna reagents ( illumina ) on a sciclone liquid handling robot ( perkinelmer ) using a perkinelmer - developed automated script . cluster generation was performed with the resulting libraries using the illumina truseq sr cluster kit v3 reagents and sequenced on the illumina hiseq 2000 using truseq sbs kit v3 reagents . a sequencing depth of 40 millions of reads using 100  bp single - end reads was used . reads were mapped to the mm9 genome with tophat ( v. 2.0.8 ) using library - type  = fr - firststrand . count data for exons were generated using htseq - count from the htseq package ( http://www.huber.embl.de/users/anders/htseq/ , version 0.5.4p3 ) using refseq annotation , with parameters stranded  = reverse and mode  = union . the normalized read counts per gene were log transformed , and then pearson correlation was used to calculate the correlation of gene expression with the physiological trait . genenetwork ( www.genenetwork.org ) variants data set comprising about 15,000 snps on the whole mouse genome was used to perform qtl mapping . calling snps on our rna - seq data have been used as a way to check for genetic drift in the bxd strains . snp calling was performed using the genome analysis toolkit from the broad institute ( v. 3.2 ) using the recommended workflow for rna - seq data . this allowed tagging the discrepant variants  compared to our rnaseq experiments  as  unknown  ( eight 0.05% genotypes per strain ) for qtl analysis . finally , variants similar to our snp calling data were merged to remove redundant information . for qtl analysis , we used the r / qtl packages with haley - knott ( hk ) regression method . qtl location was obtained with 1.5 lod ( 6.915 lrs ) support intervals as suggested previously ( broman and sen , 2009 ) . the significance and suggestive thresholds were computed with 2,000 permutations : the sequences of the various strains were shuffled 2,000 times and association recalculated at each permutation . this allowed calculating the probability to find these associations by chance . to test the efficiency of the fgf15 shrnas , an fgf15 cdna was subcloned in  frame with gfp in the pcmv6-ac - gfp plasmid ( cat . number : mg202456 , origene ) and scrambled or fgf15-targeted shrnas were subloned in the pgfp - c - shlenti plasmid ( cat . the vectors were  co - transfected into hek293 t cells using lipofectamine 3000 ( cat . number : l3000015 , thermo fisher scientific ) . at 48  hr later , total rna and  proteins were extracted and fgf15 expression was determined by qrt - pcr and protein by western blot . the selected shrna sequence has the following  sequence : 5-gtctgtgtcagatgaagatccactctttc-3. lentiviruses were produced as described ( salmon and trono , 2006 ) . the silencing efficacy of the fgf15-shrna lentivirus was tested in infected hek293 t cells that were transfected with fgf15 expression plasmid . cannulas were placed in the lateral ventricle ( 0.7  mm from the bregma ; 1.3  mm from the midline ; and 1.8  mm from the surface of the skull ) ( paxinos and watson , 1982 ) . lentiviruses ( 200 nl ; 4.10 tu / ml ) were injected in the dmh ( 1.7  mm from the bregma ;  0.35  mm from the midline ; and 5.0  mm from the surface of the skull ) and pef ( 1.7  mm from the bregma ;  0.75  mm from the midline ; and 5.15  mm from the surface of the skull ) ( paxinos and watson , 1982 ) . animals were allowed to recover for 1  week with daily handling and body weight monitoring after i.c.v . the placement of the cannula was checked by histological analysis after blue ink delivery , and the sites of injection ( gfp fluorescence ) were verified on 40  m brain sections . acsf ( 125  mm nacl , 2.5  mm kcl , 1.25  mm nah2po4 , 1  mm mgcl2 , 2  mm cacl2 , 26  mm nahco3 and 12.5  mm sucrose , 300 mosm , and ph  = 7.4 ) or fgf19 ( 2.5  g ) ( cat . number : cyt-700-b , prospecbio ) . one hr later , mice were injected i.p . with nacl 0.9% or 2dg 600  mg / kg . blood and brains were collected 30  min later for quantification of glucagon secretion or for in  situ hybridization detection of c - fos . for perk1/2 detection tissues preparation , rna extraction , and qrt - pcr were performed as described ( mounien et  al . , 2010 ) . the following forward ( f ) and reverse ( r )  primers were used : fibroblast growth factor 15 ( fgf15 ) f5-gaggaccaaaacgaacgaaatt-3 and r5-acgtccttgatggcaatcg-3 ; fibroblast growth receptor 1 isoform c ( fgfr1c ) : f5-ggaggtgcttcatctacgga-3and r5-agagtgatgggagagtccga-3 ; fibroblast growth receptor 4 ( fgfr4 ) : f5-gcccgacagttctctttgga-3 and r5- ttcccaaagcggatcgagag-3 ; klotho beta ( klotho beta ) : f5-ccttcccactggcaatctgt-3and r5-gggtggtacaacgtcaccat-3 ; and beta - glucuronidase ( gusb ) : f5-gtgatggaggagctggttcg-3 and r5-agcagaggaaggctcattgg-3. gusb was used as a housekeeping gene to normalize gene expression in all experiments . for immunofluorescence detection of perk1/2 , mice were fixed by a transcardiac perfusion of 4% cold paraformaldehyde ( pfa ) in sodium phosphate buffer ( 0.1 m , ph 7.4 ) . brains were dissected and kept for 2  hr in pfa at 4c before overnight incubation in sucrose 30% at 4c and frozen at 80c . there were 20  m cryosections that were prepared , placed on glass slides , and incubated in a blocking buffer consisting of 0.1  m phosphate buffer ph 7.4 containing 3% of normal goat serum and 0.3% triton x-100 for 1  hr , then with a rabbit monoclonal antibody directed against perk1/2 ( cat . number : 4370 , 1/200 , cell signaling ) for 48  hr , and for 1.5  hr with an alexa fluor 488-conjugated goat anti - rabbit igg ( h+l ) antibody ( cat . for in  situ hybridization analysis , the hypothalamus and brainstem were dissected using a mouse brain matrix with 1-mm section dividers ( cellpoint scientific ) ; all tissues were fixed for 28  hr in 10% formalin before embedding in paraffin . there were 5  m sections that were cut and in  situ hybridization for fgf15 , c - fos , neun , and dihydrodipicolinate reductase ( dapb ) ( cat . number : 412811 , 316921 , 313311 , and 310043 ) was processed using the rnascope and rnascope 2.0 hd red detection kit assay ( advanced cell diagnostics ) . number : 363123s , vwr ) , and observed using an axio imager d1 ( zeiss ) microscope interfaced with axiovision software ( zeiss ) . quantification of c - fos expression was performed on sections cut at bregma 7.56  mm from three different mice . the firing rates of the thoracic branch of both vagal and sympathetic nerves along the carotid artery were recorded as previously described ( magnan et  al . unipolar nerve activity was recorded continuously for 1  hr under isoflurane anesthesia ( 30  min during basal condition after i.c.v . 2dg and 30  min during 2dg - induced neuroglucopenia ) using the labchart 8 software ( ad instrument ) . statistical analysis were performed using graphpad prism 5.0c , either by a one - way anova followed by bonferroni post hoc test , a two - way anova followed by bonferroni post hoc test , or by an unpaired two - tailed student s t test when appropriate . , j.s . , and x.b . performed the phenotyping of glucagon secretion in bxd mice .
summarythe counterregulatory response to hypoglycemia , which restores normal blood glucose levels to ensure sufficient provision of glucose to the brain , is critical for survival . to discover underlying brain regulatory systems 
 , we performed a genetic screen in recombinant inbred mice for quantitative trait loci ( qtl ) controlling glucagon secretion in response to neuroglucopenia . 
 we identified a qtl on the distal part of chromosome 7 and combined this genetic information with transcriptomic analysis of hypothalami . 
 this revealed fgf15 as the strongest candidate to control the glucagon response . 
 fgf15 was expressed by neurons of the dorsomedial hypothalamus and the perifornical area . 
 intracerebroventricular injection of fgf19 , the human ortholog of fgf15 , reduced activation by neuroglucopenia of dorsal vagal complex neurons , of the parasympathetic nerve , and lowered glucagon secretion . 
 in contrast , silencing fgf15 in the dorsomedial hypothalamus increased neuroglucopenia - induced glucagon secretion . 
 these data identify hypothalamic fgf15 as a regulator of glucagon secretion .