(PDF) A neural circuit for gut-Induced reward-蚂蚁淘生物

ArticleA Neural Circuit for Gut-Induced RewardGraphical AbstractHighlightsdCritical role for the vagal gut-to-brain axis in motivation andrewarddOptogenetic stimulation of the vagal gut-to-brain axisproduces reward behaviorsdAsymmetric brain pathways of vagal origin mediatemotivation and dopamine activitydGut-innervating vagal sensory neurons are majorcomponents of the reward circuitryAuthorsWenfei Han, Luis A. Tellez,Matthew H. Perkins, ...,Sara J. Shammah-Lagnado,Guillaume de Lartigue, Ivan E. de AraujoCorrespondenceivan.dearaujo@mssm.eduIn BriefA gut-to-brain neural circuit establishesvagal neurons as an essential componentof the reward neuronal pathway, linkingsensory neurons in the upper gut tostriatal dopamine release.Han et al., 2018, Cell 175, 665–678October 18, 2018 ª2018 Elsevier Inc.https://doi.org/10.1016/j.cell.2018.08.049 ArticleA Neural Circuit for Gut-Induced RewardWenfei Han,1,2,3,12Luis A. Tellez,1,2Matthew H. Perkins,3Isaac O. Perez,1,4Taoran Qu,1Jozelia Ferreira,1,2,5Tatiana L. Ferreira,1,2,6Daniele Quinn,1Zhong-Wu Liu,7Xiao-Bing Gao,7Melanie M. Kaelberer,8Diego V. Boho´rquez,8,9Sara J. Shammah-Lagnado,10Guillaume de Lartigue,1,11and Ivan E. de Araujo1,2,3,11,12,13,*1The John B. Pierce Laboratory, New Haven, CT, USA2Department of Psychiatry, Yale University School of Medicine, New Haven, CT, USA3Fishberg Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, NY, USA4Section of Neurobiology of Oral Sensations, FES-Iztacala, National Autonomous University of Mexico, Mexico City, Mexico5Department of Anatomy, Biomedical Sciences Institute, University of Sa˜o Paulo, Sa˜o Paulo, Sa˜o Paulo, Brazil6Mathematics, Computing and Cognition Center, Federal University of ABC, Sa˜o Bernardo do Campo, Sa˜o Paulo, Brazil7Department of Comparative Medicine, Yale University School of Medicine, New Haven, CT, USA8Department of Medicine, Duke University, Durham, NC, USA9Department of Neurobiology, Duke University, Durham, NC, USA10Department of Physiology and Biophysics, Biomedical Sciences Institute, University of Sa˜o Paulo, Sa˜o Paulo, Sa˜o Paulo, Brazil11Department of Physiology, Yale University School of Medicine, New Haven, CT, USA12Present address: Fishberg Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, NY, USA13Lead Contact*Correspondence: ivan.dearaujo@mssm.eduhttps://doi.org/10.1016/j.cell.2018.08.049SUMMARYThe gut is now recognized as a major regulator ofmotivational and emotional states. However, therelevant gut-brain neuronal circuitry remains un-known. We show that optical activation of gut-innervating vagal sensory neurons recapitulatesthe hallmark effects of stimulating brain reward neu-rons. Speciﬁcally, right, but not left, vagal sensoryganglion activation sustained self-stimulationbehavior, conditioned both ﬂavor and place prefer-ences, and induced dopamine release from Substan-tia nigra. Cell-speciﬁc transneuronal tracing revealedasymmetric ascending pathways of vagal originthroughout the CNS. In particular, transneuronal la-beling identiﬁed the glutamatergic neurons of thedorsolateral parabrachial region as the obligatoryrelay linking the right vagal sensory ganglion to dopa-mine cells in Substantia nigra. Consistently, opticalactivation of parabrachio-nigral projections repli-cated the rewarding effects of right vagus excitation.Our ﬁndings establish the vagal gut-to-brain axis asan integral component of the neuronal rewardpathway. They also suggest novel vagal stimulationapproaches to affective disorders.INTRODUCTIONFor centuries, the gastrointestinal branches of the vagus nervehave been recognized as the core of the gut-brain axis (Puizillout,2005). Conventionally, the function attributed to vagal gut affer-ents is the transmission of meal-borne signals to the brain, ulti-mately acting as the chief negative-feedback mechanism formeal size regulation (Schwartz, 2000). In such classical model,gut vagal afferents sensitive to mechanical and chemical signals(Prechtl and Powley, 1990; Williams et al., 2016) act to reducefood reward by engaging their terminal ﬁelds in caudal brainstem(Norgren and Smith, 1988; Schwartz, 2000). In contrast, circu-lating hormones, rather than vagal transmission, are the factorsconventionally believed to convey gut-borne rewarding signalsto the brain (Berthoud, 2008; Sclafani, 2013).More recently, however, a wider range of neuropsychologicalprocesses has been attributed to the vagal gut-brain axis. Theseinclude anxiety, depression, cognition, and reinforcement (Bo-ho´rquez and Liddle, 2015; Clemmensen et al., 2017; de Araujoet al., 2012; Mayer, 2011; Sharon et al., 2016). Still, the neural cir-cuitry allowing gastrointestinal control over motivation andreward remains unmapped. Advances in this ﬁeld have beengreatly limited by the technical difﬁculties associated withisolating vagal afferents in an organ-speciﬁc manner (de Lartigueand Diepenbroek, 2016). To address this question, we made useof virally delivered molecular tools to specify the behavioral func-tions and central relays of gut-innervating sensory vagal affer-ents. We focused on challenging the long-held assumption thatvagal sensory neurons act to inhibit reward circuits, thereby sup-pressing motivated behavior (Angya´n, 1975).RESULTSTargeting Sensory Vagal Neurons for Gut-BrainOptogeneticsOur ﬁrst challenge was to develop an experimental preparationwhere we could separately manipulate the sensory branch ofthe vagus nerve. More speciﬁcally, we aimed at manipulatingthe excitability of upper gut-innervating sensory neurons of thevagus. We employed a combinatorial viral approach to achieveour aims. We ﬁrst transfected the stomach and duodenum ofwild-type mice with a retrogradely transported, adeno-associ-ated virus carrying a Cre-EBFP construct (AAVrg-pmSyn1-EBFP-Cre) (Tervo et al., 2016). In this way, Cre recombinaseCell 175, 665–678, October 18, 2018 ª2018 Elsevier Inc. 665 was retrogradely and bilaterally transported into the sensoryganglia of the vagus nerve (i.e., the nodose ganglia [NG]). Next,we separately transfected the right and left NG with the light-sen-sitive depolarizing channel Channelrhodopsin2 by injecting theCre-inducible viral construct AAV-EF1a-DIO-hChR2(H134R)-EYFP (ChR2) (Madisen et al., 2012). Because the expression ofthe light-sensitive channel is Cre-dependent, laser pulses wouldaffect only NG neurons expressing Cre recombinase—in thiscase, upper gut-innervating NG neurons retrogradely targetedby upper gut injections of the Cre-carrying retrograde virus.The overall viral approach is depicted in Figure 1A.Upon DIO-ChR2-EYFP injections into the right nodose gan-glion (R-NG, Figures 1A and S1A), overlapping expression ofAAVrg-pmSyn1-EBFP-Cre and DIO-ChR2-EYFP was readily de-tected in R-NG (Figures 1B and S1A). Consistent with a regionallyrestricted expression of EYFP within R-NG (Figure S1B), confocalmicroscopy of clariﬁed gut tissue revealed EYFP-positive ﬁbersinnervating the upper-gut but no other organs (Figures S1C–S1E). Nerve recordings obtained upon laser stimulation of thevagal trunk revealed robust, light-locked electrical activity acrossvagal ﬁbers (Figure 1C). Nerve responses occurred in theabsence of detectable changes in gastric motility (Figure S1F).In the CNS, ascending EYFP-positive vagal ﬁbers arising fromR-NG were concentrated in ventromedial areas of the nucleus ofthe solitary tract (NTS) (Figure 1D). In order to achieve stimulationof these gut-innervating R-NG terminals in awake behavingmice, an optic ﬁber was placed immediately above these NGneuronal terminals in medial NTS (Figure 1E). Laser pulses deliv-ered through the ﬁber signiﬁcantly increased neuronal activity inlower brainstem (Figure S1G).Optical Activation of Upper Gut-Innervating VagalSensory Neurons Induces RewardUsing a series of behavioral tests, we ﬁrst assessed the rein-forcing value associated with optically exciting the uppergut-innervating R-NG neurons. Strikingly, we ﬁrst observedthat fast optogenetic activation of R-NG terminals sustainedself-stimulation behavior, the hallmark assay for identifyingreward neurons (Olds and Milner, 1954; Schultz, 2015).Speciﬁcally, transfected mice produced increasingly greaternumbers of operant responses to obtain blue light deliveryto R-NG terminals in NTS; conditioned responses wereconsistently vigorous, including during subsequent extinctiontests (Figure 1F; Video S1).Figure 1. Gut-Brain Optogenetics(A) Combinatorial viral strategy to target gut vagal sensory neurons.(B) Cre-EBFP and DIO-ChR2-EYFP detected in R-NG. Scale bar, 100 mM.(C) Left: Vagal nerve ﬁber activity upon light stimulation of R-NG DIO-ChR2-EYFP-positive neurons. Trace shows responses locked to light pulses (blue bars ontop). Right: Supra-threshold events during nerve recordings. n = 12; paired t test t[11] = 8.7 *p 0.001.(D) R-NG DIO-ChR2-EYFP-positive terminals innervate ventromedial NTS at both anterior (i) and caudal (ii) levels (103). Lower panels show delimited areas at403. AP (a and c) innervation was weaker than NTS (b and d) at both anterior (i) and caudal (ii) levels. Scale bar, 100 mM.(E) Optical ﬁbers above R-NG terminals in NTS.(F) R-NG /NTS optical activation sustains self-stimulation. N = 6; two-way RM-ANOVA, main effect of poking on the laser-paired hole versus inactive holeF[1,5] = 60.4, p = 0.001. Mice gradually increased the number of responses over daily sessions: main effect of session, F[2,10] = 8.0, p = 0.008. Increases inresponse rates were speciﬁc to poking on the laser-paired hole: laser 3session interaction effect: F[2,10] = 13.7, *p = 0.001. During laser-off extinction tests, micepoked signiﬁcantly more on the laser-paired hole, paired t test t[5] = 16.3, **p 0.001.(G) R-NG /NTS optical stimulation induces place preferences. Left: Representative heatmap showing the pre-test baseline (upper) and on-line place preference(lower). Right: Place preference for laser-paired side, N = 6; paired t test t[5] = 7.2, *p = 0.001.(H) R-NG /NTS optical stimulation induces ﬂavor preferences. Post-conditioning ﬂavor preferences for laser-paired ﬂavors N = 6; paired t test t[5] = 14.8, *p 0.001. For one-sample t tests against 50% (indifference) preferences: pre-conditioning: t[5] = 0.1, p = 0.89; post-conditioning, t[5] = 10.8 Bonferroni-correctedp 0.0001.(I) R-NG /NTS optical stimulation reduces chow intake. N = 5, three daily sessions (laser on day 2), one-way RM-ANOVA F[2,8] = 34.6, *p 0.0001).(J) Optical stimulation of R-NG /NTS, but not R-NG /AP, induced robust satiety during 5% IntraLipid intake tests. Shaded blue area indicates sessions whenlaser was ON. N = 5, two-way mixed effects ANOVA, group 3session interaction: F[8,64] = 7.8, *p 0.001.(K) Optical stimulation of R-NG /NTS, but not R-NG /AP, ﬁbers induced robust dopamine release in dorsal striatum. Shaded blue area indicates sessionswhen laser was ON. N = 5, two-way mixed effects ANOVA between-group 3time interaction effect F[15,120] = 3.4, *p 0.0001.(L) Cre-EBFP and DIO-ChR2-EYFP detected in L-NG. Scale bar, 100 mM.(M) Left: Vagal nerve ﬁber activity upon light stimulation of L-NG DIO-ChR2-EYFP-positive neurons. Responses were mostly locked to light pulses (blue bars ontop). Right: Supra-threshold events during nerve recordings. n = 12; paired t test t[11] = 4.7, *p = 0.001.(N) L-NG DIO-ChR2-EYFP-positive terminals substantially innervated AP at more caudal (i) but not anterior (ii) levels (103). Lower panels show the delimited areasat 403. NTS (b and d) innervation was weaker than AP (a and c) at more caudal (i) but not anterior (ii) levels. Scale bar, 100 mM.(O) Optical ﬁbers above L-NG terminals in AP.(P) L-NG /AP optical stimulation failed to sustain self-stimulation. N = 5; two-way RM-ANOVA, laser 3session interaction effect: F[2,8] = 0.7, p = 0.5. Duringlaser-off extinction tests, mice failed to poke more on the laser-paired hole, paired t test t[4] = 0.1, p = 0.8.(Q and R) L-NG /AP optical stimulation failed to induce online place preferences (laser paired to less preferred location, t[4] = 1.2, p = 0.3, (Q) oraversions (laserpaired to more preferred location, t[4] = 2.0, p = 0.1) (R).(S) L-NG /AP optical stimulation reduces chow intake. N = 5, three daily sessions (laser on day 2), one-way RM-ANOVA F[2,8] = 16.5, *p = 0.001).(T) Optical stimulation of L-NG /AP, but not L-NG /NTS, induced satiety during 5% IntraLipid intake. Shaded blue area = laser ON. N = 5, two-way mixedeffects ANOVA, group 3session interaction: F[8,64] = 18.6, *p 0.001.(U) Optical stimulation of neither L-NG /AP nor L-NG /NTS induced dopamine release in DS. Shaded blue area = laser ON. N = 5, two-way mixed effectsANOVA between-group 3time interaction F[15,120] = 0.8, p = 0.6. AP, area postrema; DMV, dorsal motor nucleus of vagus; NTS, nucleus of the tractus solitarius.Data reported as mean ±SEM.See also Figure S1 and Video S1.Cell 175, 665–678, October 18, 2018 667 During ‘‘on-line’’ place preference tests (in which the laserswitch is instantaneously controlled by the animal’s current po-sition), mice mostly stayed at those cage locations paired to laseractivation (Figure 1G). Similarly, in ﬂavor conditioning assays,robust preferences were displayed for those initially insipid,non-caloric ﬂavors that had been paired to laser activation (Fig-ures 1H and S1H). The same optical stimulation protocolinduced signiﬁcant reductions in chow (Figure 1I) and lipid (Fig-ures 1J and S1I) intake, demonstrating that food reward is notmutually exclusive to satiation. None of the light-induced effectsdescribed above were observed in mice not expressing the op-togenetic construct in R-NG (Figures S1J–S1R).Optical Activation of Upper Gut-Innervating VagalSensory Neurons Induces Dopamine Release in BrainReward PathwaysDopamine release from Substantia nigra,pars compacta (SNc)onto dorsal striatum (DS) is required for behavioral reinforcementby natural rewards as well as by stimulation of brain reward neu-rons (Crow, 1971; Palmiter, 2008; Phillips et al., 1976; Unger-stedt, 1971). Accordingly, we inquired whether remote excitationof vagal sensory neurons is sufﬁcient to induce the release ofdopamine from nigral dopamine neurons onto DS. Using micro-dialysis sampling coupled to electrochemical detection, wefound that optically exciting the upper gut-innervating R-NGneurons signiﬁcantly increased dopamine levels in DS (Fig-ure 1K), consistent with a role for gut-innervating R-NG neuronsin reward. Accordingly, R-NG stimulation modulated neuronalensemble activity in DS (Figures S1S–S1V).In sum, R-NG neurons appear to function similarly to brainreward neurons, namely, R-NG activation sustains self-stimula-tion, conditions stimulus preferences, and excites brain dopa-mine cells (Olds, 1976; Schultz, 2015).Laser-Induced Rewarding and Dopaminergic EffectsAre Speciﬁc to the R-NGIn animals similarly injected with the ChR2 construct in the leftnodose ganglion (L-NG), overlapping expression of AAVrg-pmSyn1-EBFP-Cre and DIO-ChR2-EYFP was equally detectedin L-NG neurons (Figure 1L). Counts of Cre-transfected cellswere comparable in R-NG versus L-NG (Figure S1A). Regionallyrestricted expression of EYFP within L-NG (Figure S1W) wasconsistent with EYFP-positive ﬁbers innervating the upper-gutbut not other organs (Figures S1X–S1Z). Left nerve recordingsalso revealed light-locked electrical activity in vagal ﬁbers(Figure 1M) independently of changes in gastric motility(Figure S1AA).Right versus Left NG Modulate Distinct BrainstemSectorsWe observed conspicuous asymmetries in NG terminal ﬁelds inthe CNS. Speciﬁcally, L-NG projects strongly to a posterior partof area postrema (AP) and lightly to the NTS (Figure 1N). This is incontrast to R-NG, which projects very substantially to the ventro-medial NTS and only lightly to posterior AP (Figure 1D). Accord-ingly, an optic ﬁber was placed immediately above these L-NGneuronal terminals in AP (Figure 1O). Optical activation of theseterminals completely failed to sustain self-stimulation behavior(Figure 1P). Similarly, neither attraction nor aversion was formedupon laser stimulation during on-line place preference tests (Fig-ures 1Q and 1R). Despite the lack of reinforcement effects, L-NGoptical stimulation did induce signiﬁcant decreases in both chow(Figure 1S) and lipid (Figure 1T) intake. Consistent with the induc-tion of satiation in the absence of rewarding effects, L-NG opticalstimulation equally failed to alter striatal dopamine levels (Fig-ure 1U). Overall, these results indicate that right, but not left,NG neurons have privileged access to Substantia nigra dopami-nergic reward neurons.To conﬁrm such functional asymmetries, we implanted opticalﬁbers above the AP (in the area innervated by L-NG terminals) inmice injected with ChR2 into the R-NG; conversely, we also im-planted optical ﬁbers above the NTS (in the area more exten-sively innervated by R-NG terminals) in mice injected withChR2 into the L-NG. In both cases neither behavioral nor dopa-minergic effects were detected (Figures S1BB–S1MM). Theseresults were paralleled by Fos expression analyses (Figure S1G).Targeted Nodose Terminal Fields Were Gut SpeciﬁcTo assess the organ-speciﬁcity of the gut terminal ﬁeldsthroughout the solitary complex, we injected a ﬁxed volume ofthe retrograde Cre-carrying construct AAVrg-pmSyn1-EBFP-Cre into the upper gut, the heart, the lung, or the trachea. In allcases, Cre-inducible DIO-ChR2-EYFP was injected into NGbilaterally. Analyses revealed a distinct organ-speciﬁc mapwhere only gut terminals innervated the ventromedial NTS (Fig-ures 2A–D and S2A–S2D). This is in agreement with earlier heartand lung terminal maps (Corbett et al., 2005; McGovern et al.,2015). Accordingly, and unlike NG neurons innervating organssuch as the lung or heart (Chang et al., 2015), gut-speciﬁc acti-vation of neither R-NG nor L-NG caused changes in respiratoryor cardiovascular tone (Figures S2E–S2H). Conversely, stimula-tion of gut-innervating parasympathetic vagal motor neuronsinduced gut motility in the absence of any rewarding or dopami-nergic effects (Figures S2I–S2M).Reward Remains Unaltered upon SimultaneousActivation of Right and Left NGPhysiological situations such as feeding may bilaterally stimulategut terminal ﬁelds. This raises the question of whether L-NGactivation alters R-NG-induced reward. While it is unfeasible totarget both R-NG and L-NG terminals using optical ﬁbers, bilat-eral nodose activation may be achieved via chemogenetics(Sternson and Roth, 2014). Accordingly, the stomach and duo-denum of wild-type mice were transfected with a retrogradelytransported, Cre-carrying construct (CAV2-Cre-GFP) (Junyentand Kremer, 2015). Then, the Cre-inducible, Gs-coupleddesigner receptor encoded in the construct AAV-hSyn-HA-rM3D(Gs)-IRES-mCherry (Farrell et al., 2013) was bilaterallyinjected into the NG of these same mice. Employing the Gs-coupled construct is justiﬁed by the robust cyclic AMP(cAMP)-induced enhancement of NG neuronal excitability (In-gram and Williams, 1994). This approach allowed for bilateral,upper gut-exclusive excitation of NG neurons and their brain-stem targets (Figures 2E, 2F, and S2N–S2Q).Administering the designer drug clozapine-N-oxide (CNO)induced the formation of robust place and ﬂavor preferences668 Cell 175, 665–678, October 18, 2018 (Figures 2G, 2H, and S2R), reductions in food intake (Figures S2Sand S2T) and increases in striatal dopamine (Figures 2I, S2U, andS2V). Thus, activating L-NG does not disrupt the rewarding anddopaminergic effects associated with activating R-NG. None ofthese effects were observed when either CNO or clozapinewere injected in mice not carrying the chemogenetic construct(Figures S2W–S2AA).A Polysynaptic Neuronal Pathway Links the Right VagalSensory Ganglion to Dopamine CellsWe next aimed at mapping the central pathways linking vagalsensory ganglia to nigral dopamine neurons. Because NG donot contact the midbrain directly, we employed an anterogradetranssynaptic viral tracer for mapping vagal outputs. BecauseNG neurons unconditionally express VGlut2 (Williams et al.,2016), we injected the Cre-inducible, transsynaptic herpes sim-plex viruses 1 strain H129DTK-TT (Lo and Anderson, 2011) intothe R-NG of VGlut2-ires-Cre mice (Figure 3A).Forty-eight hours after the injections, infection was restrictedto the dorsal vagal complex, including NTS (Figures 3B and3C). This pattern was consistent with the ones obtained frominjecting the Cre-inducible monosynaptic synaptophysinconstruct AAV8.2-hEF1a-DIO-synaptophysin-EYFP into theR-NG of VGlut2-ires-Cre mice (Figure S3A). Seventy-two hourspost-injection, infection extended onto NTS targets, includingparaventricular hypothalamic nuclei (Figure 3D), in addition tothe dorsolateral region of the parabrachial nucleus (PBNdl) (Fig-ures 3E and S3B). Ninety-six hours post-injection, a number ofregions rostral to PBNdl were infected including the lateralaspect of SNc (Figure 3F). Several infected neurons in SNcwere found to be dopaminergic (Figure 3G). A detailed descrip-tion of infection patterns up to 120 hr post-injection is provided inFigure S3B.The PBNdl Links the Right Vagal Sensory Ganglion toSubstantia nigraConsistent with previous reports (Coizet et al., 2010), injection ofa retrograde dye into SNc predominantly revealed profuse label-ing in PBNdl (Figure S3C). Critically, labeling was restricted to thesite where herpes infection was observed (excluding thereforeFigure 2. Organ-Speciﬁc Maps of Gut Vagal Afferents(A–D) The retrograde Cre-carrying construct AAVrg-pmSyn1-EBFP-Cre was injected into upper gut, heart, lung, or trachea. In all cases, DIO-ChR2-EYFP wasbilaterally injected into NG. Gut (A), heart (B), trachea (C), and lung (D) terminal sites were restricted to distinctively separate sites within the solitary complex.Additional sections shown in Figures S2A–S2D.(E) The upper gut of mice was injected with the retrograde construct CAV2-Cre-GFP, and NG were bilaterally injected with a Cre-inducible AAV-Gs-coupled-mCherry chemogenetic designer receptor construct. CAV2-Cre-GFP and AAV-DIO-Gs-mCherry were detected in NG bilaterally. Middle: mCherry expression isrestricted to outermost nodose neurons (103, scale bar, 100 mM). Right: 403, scale bar, 100 mM.(F) AAV-DIO-Gs-mCherry-infected terminals by injections into both right nodose (terminal ﬁbers in medial NTS) and left nodose (terminal ﬁbers in AP).(G) CNO injections induce place preferences. During conditioning, in all sessions animals were administered an intragastric infusion (I.G.) with IntraLipid (0.3 kcal,0.6 mL). CNO injections were then paired to the less preferred side of the cage. Left: Representative heatmap showing the pre-test baseline (upper) and post-CNO preferences (lower). Right: Place preference for CNO-paired side, N = 5; paired t test t[4] = 9.2, *p = 0.001.(H) Post-conditioning ﬂavor preferences for ﬂavors paired with 5% I.G. IntraLipid (0.3 kcal, 0.6 mL) + CNO versus 5% I.G. IntraLipid + saline, N = 5; paired t test t[4] = 9.5, *p = 0.001. For one-sample t tests against 50% (indifference) preferences: pre-conditioning: t[4] = 1.6, p = 0.18; post-conditioning, t[4] = 7.5, Bonferroni-corrected p = 0.004.(I) CNO injections signiﬁcantly enhanced dopamine release in dorsal striatum induced by 5% I.G. IntraLipid (0.3 kcal, 0.6 mL), N = 6; two-way RM-ANOVA CNO 3sampling time interaction effect F[18,72] = 7.59, *p 0.0001. AP, area postrema; DMV, dorsal motor nucleus of vagus; NTS, nucleus of the tractus solitarius. Datareported as mean ±SEM.Cell 175, 665–678, October 18, 2018 669 Figure 3. Transsynaptic Labeling of Central Vagal Pathways(A) VGlut2-ires-Cre mice were injected with the Cre-inducible, transsynaptic herpes simplex viruses 1 strain H129DTK-TT into the right R-NG.(B and C) 48 hr after the injections, infection was restricted to the dorsal vagal complex (AP, DMV, NTS) at more caudal (B) and rostral (C) levels.(D and E) 72 hr after injection infection was detected at NTS targets including PVH (D) and PBNdl (E).(F) 96 hr after injections infection was detected in SNc, including in dopaminergic, tyrosine hydroxylase-positive (TH, green) cells.(G) Numbers of herpes-infected cells in SNc and VTA, including both GABAergic and TH+cells (values correspond to average numbers of infected cells persection, N = 3 mice).(H–K) The L-NG was injected with H129DTK-TT. Infection was observed in the dorsal vagal complex (AP, DMV, NTS) (H), PVH (J), PBNel (I). Note absence ofherpes labeling in PBNdl (I), VTA and SNc (K).(L) Upper gut transfected with AAVrg-pmSyn1-EBFP-Cre, R-NG then injected with H129DTK-TT.(legend continued on next page)670 Cell 175, 665–678, October 18, 2018 the externolateral parabrachial nucleus [PBNel]) (Figure S3C). Noretrograde labeling was detected in the dorsal vagal complex.We thus hypothesized that PBNdl links the vagal central path-ways to SNc. Because VGlut2 is enriched in the lateral para-brachium (Kaur et al., 2013), we more speciﬁcally predictedthat vagal sensory information reaches SNc via an excitatoryrelay in PBNdl. We introduced the viral construct AAV-ﬂex-ta-Casp3-TEVp (Yang et al., 2013), which induces Cre-dependentcaspase expression, bilaterally into the PBNdl of VGlut2-ires-Cre mice. After 3 weeks, we injected the R-NG of these samemice with the H129DTK-TT construct. Despite infection of thedorsal vagal complex and their hypothalamic targets, labelingwas completely abolished in both PBNdl and SNc (Figure S3D).Remarkably, H129DTK-TT injections into the L-NG produced nolabeling in either PBNdl or SNc (Figures 3H–3K and S3E). Thelatter is consistent with the optogenetic data revealing R-NG-speciﬁc effects.The Source of Vagal Sensory Inputs to Dopamine Cells Isthe Upper GutWe next aimed at determining whether gut-innervating nodoseneurons are necessary and sufﬁcient sources of vagal inputs toSNc. We again transfected the stomach and duodenum ofwild-type mice with the retrograde construct AAVrg-pmSyn1-EBFP-Cre, and after 3 weeks H129DTK-TT was injected in theR-NG of these mice (Figure 3L). We again observed infectionof both PBNdl and lateral SNc dopaminergic cells (Figures 4M–4R and S4F), demonstrating that transsynaptic labeling fromupper gut-innervating R-NG neurons is sufﬁcient to induce label-ing in SNc.Conversely, we also induced selective vagal deafferentation ofthe gut previous to introducing the transsynaptic virus into R-NG.This was achieved by injecting the neurotoxin saporin conju-gated to cholecystokinin (CCK-SAP) (Diepenbroek et al., 2017)into the R-NG of VGlut2-ires-Cre mice. The R-NG of thesemice was then injected with H129DTK-TT. After vagal deafferen-tation of the gut, labeling was restricted to the dorsal motor nu-cleus of the vagus (Figure S3G).Separate Regions of the Lateral Parabrachial NucleusRespond to Aversive versus Rewarding StimuliThe lateral parabrachial region is known to mediate avoidancebehaviors, including responses to visceral malaise (Carteret al., 2013). This is in principle inconsistent with our results sug-gesting a lateral parabrachial relay for vagus-induced reward.This apparent contradiction led us to hypothesize that the lateralparabrachial complex contains separate subnuclei that act tomediate reward versus avoidance behaviors.We used Fos induction to map lateral parabrachial responsesto aversive versus rewarding visceral stimuli. Speciﬁcally, wecompared Fos responses to systemic injections of the vagus-mediated digestive peptide cholecystokinin (CCK) (Smith et al.,1985) versus to malaise-inducing lithium chloride (LiCl). Impor-tantly, CCK and LiCl doses were titrated so that both treatmentsproduced a statistically equivalent, 50% reduction in foodintake (Figure 4A). Despite producing the same behavioraloutcome, injections of these doses of CCK and LiCl inducedhighly dissimilar Fos expression patterns in the lateral para-brachium: whereas LiCl responses were restricted to the exter-nolateral aspect (PBNel), CCK responses were circumscribedto dorsolateral and, to a lesser extent, medial regions (PBNdland PBNm) (Figures 4B–4E). Thus, neuronal responses toCCK, but not to LiCl, overlapped with the herpes-infected rightvagus-recipient parabrachial areas.CGRP-positive neurons of the lateral parabrachium are criticalmediators of avoidance behaviors (Carter et al., 2013). We there-fore hypothesized that Fos induced by LiCl, but not by CCK,overlaps with CGRP expression. Double immunostainingconﬁrmed our assumption that LiCl-responsive neurons amal-gamate with CGRP-positive neurons within PBNel, whereasmost CCK-responsive neurons are CGRP-negative cells locatedmore dorsally within PBNdl (Figures 4F, 4G, and S4A–S4F).Vagus-Recipient Parabrachial Regions PreferentiallyTarget Substantia nigraBased on the above, we predicted that PBNdl and PBNel shouldsend their efferents to separate downstream targets. To obtainsmall injections restricted to parabrachial subnuclei, we injectedthe sensitive anterograde tracer Phaseolus vulgaris leucoagglu-tinin (PHA-L) into PBNdl versus PBNel.We found that PHA-L injections restricted to PBNdl—whereboth CCK-responsive and R-NG-transsynaptic targets arelocated (Figure 3E)—resulted in a dense terminal ﬁeld in themidbrain dopaminergic complex, including SNc (Figures 4H,4I, S4H, and S4I). Importantly, concomitant injections of theretrograde tracer FluoroGold into DS (Figure S4G) revealedthat PBNdl terminal ﬁbers and DS-projecting neurons are in reg-ister in SNc (Figure 4J), suggesting the existence of a parabra-chio-nigro-striatal pathway.Strikingly, PHA-L injections into PBNel resulted in virtually nolabeling in dopaminergic midbrain regions (Figures 4K andS4J). In contrast, these PBNel injections resulted in a dense ter-minal ﬁeld in the caudal lateral and capsular parts of the centralamygdaloid nucleus (CeL/C) (Figure S4K). Consistently, retro-grade tracer injections into CeL/C resulted in a robust labelingthat was restricted to PBNel (Figures S4L and S4M). Accord-ingly, Fos responses to LiCl were restricted to the amygdalartargets of PBNel (Figure S4N).To assess the cell-type speciﬁcityof parabrachio-nigraltargets,we transfected the SNc of both DAT-ires-Cre and VGat-ires-Cremice with the Cre-inducible, retrograde pseudotyped monosyn-aptic rabies virus SADDG-GFP (Figure 4L) (Wickersham et al.,(M–Q) Infection was observed in the dorsal vagal complex (AP, DMV, NTS) at more caudal (M) and rostral (N) levels, PVH (O), PBNdl (P), PBNel (P), and SNc (Q).(R) Numbers of herpes-infected cells in SNc and VTA, including both GABAergic and TH+cells (values correspond to average numbers of infected cells persection, N = 3 mice, main effect of cell type F[1,2] = 28.76, *p = 0.03). AP, area postrema; DMV, dorsal motor nucleus of the vagus; NTS, nucleus of the solitarytract; PBNdl, parabrachial nucleus, dorsolateral region; PBNel, parabrachial nucleus, externolateral part; PVH, paraventricular nucleus of the hypothalamus; SNc,Substantia nigra,pars compacta; VTA, ventral tegmental area. Scale bars, 100 mM. Data reported as mean ±SEM.See also Figure S3.Cell 175, 665–678, October 18, 2018 671 Figure 4. Valence-Speciﬁc Organization of the Lateral Parabrachial Area(A) Intraperitoneal doses of CCK and LiCl, were titrated to induce 50% reduction in food intake. Main effect of treatment F[2,12] = 38.65 p 0.001. N = 5; Pairedt test saline versus CCK Bonferroni p 0.001; saline versus LiCl Bonferroni p 0.001; CCK versus LiCl p = 0.8.(B) The mouse parabrachial area. PBNdl, parabrachial nucleus, dorsolateral region; PBNel, externolateral parabrachial nucleus; PBNm, medial parabrachialnucleus; scp, superior cerebellar peduncle (brachium conjunctivum).(C and D) Compounded image of parabrachial sections showing Fos expression patterns in response to CCK (C) and LiCl (D) across 5 mice.(E) Fos+cells in response to CCK or LiCl. PBNdl speciﬁcally responded to CCK, whereas PBNel speciﬁcally responded to LiCl. No responses for saline injections.N = 5 in each group. Main effect of treatment in PBNdl: F[2,12] = 21.9, p 0.0001; post hoc two-sample t tests saline versus LiCl p = 0.249; saline versus CCKBonferroni *p 0.001; LiCl versus CCK Bonferroni **p = 0.002. Main effect of treatment in PBNel: F[2,12] = 59.5, p 0.0001; post hoc two-sample t tests salineversus LiCl Bonferroni *p 0.001; saline versus CCK p = 0.9; LiCl versus CCK Bonferroni **p 0.001. Main effect of treatment in medial PBN (PBNm): F[2,12] = 0.8,p = 0.471; Post hoc two-sample t tests all p 0.85.(F) No Fos expression in response to CCK was observed in PBNel (left), in particular no CGRP+neurons (center) were found to express Fos in response toCCK (right).(G) Robust Fos expression in response to LiCl was observed in PBNel (left). The region containing CGRP+neurons (center) was found to include most of the LiCl-responding Fos-positive neurons (right).(H) The anterograde tracer PHA-L was iontophoretically injected into either PBNdl or PBNel; in the same PBNdl case the retrograde tracer FluoroGold wasiontophoretically injected into the dorsal striatum.(I) Dark-ﬁeld photomicrographs of coronal sections revealing a substantial anterograde labeling in the SNc after an injection in PBNdl. Note that these projectionpatterns were highly speciﬁc, with the Substantia nigra,pars reticulata (SNr) devoid of parabrachial terminals. Scale bar, 100 mm.(J) Bright-ﬁeld photomicrograph showing PHA-L anterograde labeling from PBNdl in register with FluoroGold retrograde labeling from the dorsal striatum in theSNc, suggesting the existence of a parabrachio-nigro-striatal pathway. Scale bar, 20 mm.(K) Fibers of passage running dorsal to the SNc, itself unlabeled, after an injection in the PBNel. Scale bar, 100 mm.(legend continued on next page)672 Cell 175, 665–678, October 18, 2018 2007). We observed dense, rabies-infected ﬁelds in PBNdl, butnot in the PBNel, of both DAT-ires-Cre and VGat-ires-Cre mice(Figures 4M and 4N). The rabies retrograde patterns furtherdemonstrate that CGRP-positive neurons (mostly restricted toPBNel) do not project to SNc (Figures 4M and 4N). No retrograderabies infection was observed in the dorsal vagal complex, con-ﬁrming that the latter does not relay vagal afferents directly toSNc (Figures S4O–S4T). In contrast, SADDG-GFP injected intothe PBNdl of VGlut2-ires-Cre mice revealed dense retrograde la-beling in the NTS ﬁeld innervated by R-NG afferents (Figures S4Uand S4V). Overall, these results support a circuit model in whichVGlut2-positive neuronsin PBNdl act as an obligatory relay linkingright nodose gut signals and SNc. Of note, AgRP-positive terminalﬁelds were detected throughout both LiCl- and CCK-responsiveareas (Figure S4W). This may account for how GABAergic AgRPneurons counteract the inhibitory actions of both CCK and LiCl(Wu et al., 2012). Finally, the anatomical ﬁndings were mirroredby functional studies revealing opposing behavioral and dopa-mine responses to CCK versus LiCl (Figures 4O–4S).Remote Stimulation of the Parabrachio-Nigral PathwayInduces Reward and Dopamine ReleaseThe labeling studies above suggest that parabrachio-nigral-, butnot parabrachio-amygdalar-, projecting neurons mediate therewarding and dopaminergic effects of vagal excitation. If ourmodel is correct, activation of PBNdl /SNc terminals shouldrecapitulate the effects observed upon remote stimulation ofgut-innervating R-NG neurons. In contrast, activation ofPBNel /CeL/C-terminals should elicit avoidance behavior.Injection of AAV8.2-hEF1a-DIO-synaptophysin-EYFP into thePBNdl of VGlut2-ires-Cre mice revealed a substantial terminalﬁeld in SNc, especially in the vagus-targeted lateral SNc (Fig-ure 5C). Slice electrophysiological recordings conﬁrmed robustexcitation of PBNdl VGlut2-neurons by blue light pulses upon in-jection of Cre-inducible Channelrhodopsin2 into the PBNdl ofVGlut2-ires-Cre mice (Figures 5B, 5D, and 5E).Optic ﬁbers were then placed above PBNdl neuronal terminalsin SNc (Figure 5A). In striking similarity to optogenetically excitingR-NG terminals, PBNdl /SNc activation sustained self-stimu-lation behavior at comparable response levels (Figure 5F; VideoS2). Likewise the gut-to-brain optogenetics results, transfectedmice produced increasingly greater numbers of operantresponses to obtain blue light delivery to SNc; conditioned re-sponses were consistently vigorous, including during subse-quent laser-off extinction tests.Additionally, during ‘‘on-line’’ place preference tests (in whichthe laser switch is instantaneously controlled by the animal’scurrent position), mice mostly stayed at those cage locationspaired to laser activation (Figure 5G; Video S3). Similarly, in ﬂavorconditioning preference tests, robust preferences were dis-played for those initially insipid, non-caloric ﬂavors that hadbeen paired to laser activation (Figures 5H and S5A). Finally,PBNdl /SNc optical activation induced robust satiety duringboth chow and lipid intake (Figures 5I, S5B, and S5C).Interestingly, injection of the Cre-inducible monosynaptic syn-aptophysin construct into PBNdl of VGat-ires-Cre mice failed tolabel terminal ﬁelds in SNc. On the other hand, similar injectionsinto the PBNel of VGat-ires-Cre mice revealed a denseGABAergic termination ﬁeld in PBNdl (Figures S5D–S5F). Opticalstimulation of these VGat-neurons, which do not project to SNc(Figures S5G and S5H), produced strong place avoidance duringon-line place preference tests as well reductions in intake(Figures S5I and S5J), further demonstrating that the rewardingeffects of stimulating PBNdl are mediated by glutamatergic pro-jections to SNc.Consistent with the effects of stimulating right vagal sensoryneurons, optical PBNdl /SNc activation induced the dopaminerelease onto DS (Figure 5J). Altogether, these results indicatethat optically activating PBNdl /SNc terminals recapitulatethe dopaminergic and behavioral effects of optically activatingR-NG neurons. In contrast, we examined the effects associatedwith stimulating the PBNel /CeL/C pathway (Carter et al.,2013). In a pattern that was strikingly opposed to PBNdl /SNc, PBNel /CeL/C optogenetic activation produced placeand ﬂavor avoidance (Figures S5K–S5R; Video S4).The Right-Vagus-parabrachio-nigrostriatal Pathway IsRequired for Nutrient SensingWe conﬁrmed the existence of a right-vagus-parabrachio-ni-grostriatal pathway by injecting a retrogradely transported,polysynaptic pseudo-rabies viral construct (Banﬁeld et al.,2003) bilaterally into DS (Figure 6A). We observed dense label-ing in the right, but not left, NG (Figures 6B and 6C). Retro-gradely labeled brain regions included the medial NTS andPBNdl upper gut terminal sites (Figures 6D–6F and S6A–S6E). Accordingly, behavioral responses to CCK were equallyabolished by striatal dopamine receptor antagonism (Fig-ure 6G), right, but not left, gut vagal deafferentation (Fig-ure 6H), and targeted lesions to the parabrachio-nigralpathway (Figure 6I). The behavioral ﬁndings were mirroredby Fos expression patterns (Figures 6J–6L). A series of loss-of-function studies demonstrate that vagal and parabrachio-nigral ablations abolish reward and dopamine release whilepreserving aversion (Figures S6F–S6LL).(L) To assess the cell-type speciﬁcity of the SNc targets of parabrachial projections, we transfected the SNc of both DAT-ires-Cre and VGat-ires-Cre mice with theconstruct AAV5-EF1a-FLEX-TVAmCherry and 2 weeks after with the Cre-inducible retrogradely transported pseudotyped rabies virus SADDG-GFP.(M and N) In both DAT-ires-Cre (M) and VGat-ires-Cre (N) mice, robust expression of rabies-infected cells were observed in PBNdl and to a lesser extent in PBNmon 7 days post rabies infection, but virtually no rabies-infected cells were observed in PBNel, including regions containing CGRP+cells. Scale bars, 100 mM.(O) Open-ﬁeld heatmaps after intraperitoneal injections of saline (upper), CCK (center), or LiCl (lower).(P and Q) LiCl injections signiﬁcantly reduced distance traveled (P) and velocity (Q) compared to both saline and CCK. CCK produced no signiﬁcant effects oneither. ANOVA F[2,12] = 15.9, p = 0.001 (distance), F[2,12] = 16.5, p 0.001 (velocity). Post hoc t tests Bonferroni *p 0.05.(R) CCK injections signiﬁcantly enhanced dopamine release in dorsal striatum N = 5; two-way RM-ANOVA injection 3sampling time interaction effectF[13,52] = 5.8, *p 0.0001.(S) LiCl injections signiﬁcantly inhibited dopamine levels in dorsal striatum N = 5; F[13,52] = 29.7 *p 0.0001. Data reported as mean ±SEM.See also Figure S4.Cell 175, 665–678, October 18, 2018 673 Figure 5. Optical Activation of Parabrachio-nigral versus Parabrachio-amygdalar Pathways Mediate Reward versus Avoidance Behaviors(A) DIO-ChR2-EYFP was injected into PBNdl of VGlut2-ires-Cre mice, and optical ﬁbers placed above parabrachial terminals on SNc (PBNdl[VGlut2] /SNcpathway). Microdialysis cannula were implanted into the dorsal striatum.(B) Injection of the DIO-ChR2-EYFP construct was restricted to PBNdl.(C) Similar injections of Cre-inducible synaptophysin-EYFP into the PBNdl of VGlut2-ires- Cre mice reveal dense glutamatergic parabrachial terminals in SNc andRRF (magniﬁcation shown on panels Ca,Cb).(D) Action potentials (current clamp) from ChR2-expressing VGlut2 neurons in PBNdl upon optogenetic stimulation.(E) Inward membrane current recorded in VGlut2-ChR2 neurons under voltage clamp. Blue bar, the applica tion of the LED-generated blue light pulse.(F) PBNdl[VGlut2] /SNc optical stimulation sustains self-stimulation behavior. N = 6; two-way RM-ANOVA, main effect of poking on the laser-paired hole versusinactive hole F[1,5] = 88.0, p 0.001. Mice gradually increased the number of responses over daily sessions: main effect of session, F[2,10] = 62.8, p 0.001.Increases in response rates were speciﬁc to poking on the laser-paired hole: laser 3session interaction effect: F[2,10] = 69.5, *p 0.001. During laser-offextinction tests, mice poked signiﬁcantly more on the laser-paired hole, paired t test t[5] = 6.589, **p = 0.001.(G) PBNdl[VGlut2] /SNc optical stimulation induces place preferences. The laser source was switched ON whenever the mouse was detected on the lesspreferred side of the cage. Left: representative heatmap showing the pre-test baseline (upper) and on-line place preference (lower). Right: place preference forlaser-paired side, N = 6; paired t test t[5] = 9.5, *p = 0.001.(H) PBNdl[VGlut2] /SNc optical stimulation induces ﬂavor preferences. Post-conditioning ﬂavor preferences for laser-paired ﬂavors N = 6; paired t test t[5] =14.6, *p 0.001. For one-sample t tests against 50% (indifference) preferences: pre-conditioning: t[5] = 1.8, p = 0.127; post-conditioning, t[5] = 5.9, Bonferroni-corrected p = 0.002.(I) PBNdl[VGlut2] /SNc optical stimulation during ingestion of 5% IntraLipid. After daily baseline sessions 1–3, intake is reduced during laser ON sessions 4–6,and immediately returned to baseline on post-laser sessions 7–9. N = 6, two-way RM-ANOVA laser 3session interaction effect F[8,40] = 9.4, *p 0.001.(J) PBNdl[VGlut2] /SNc optical stimulation induces signiﬁcant dopamine release in dorsal striatum, N = 6, two-way RM-ANOVA laser 3samplin g timeinteraction effect F[15,60] = 7.1, *p 0.0001. PBNdl, parabrachial nucleus, dorsolateral part; SNc, Substantia nigra,pars compacta; RRF, retrorubral ﬁeld. Scalebars, 100 mM. Data reported as mean ±SEM.See also Figure S5 and Videos S2,S3, and S4.674 Cell 175, 665–678, October 18, 2018 DISCUSSIONOur ﬁndings reveal the existence of a neuronal population of‘‘reward neurons’’ amid the sensory cells of the right vagusnerve. Formally, these vagal sensory neurons operate underthe same constraints attributed to reward neurons of the CNS(Olds, 1976; Schultz, 2015). Thus, gut-innervating R-NG neuronslink peripheral sensory cells to the previously mapped popula-tions of reward neurons in brain (Crow, 1973; Olds, 1976;Schultz, 2015).Our ﬁndings more speciﬁcally imply that food reinforcementand satiation should not be considered mutually exclusivephysiological processes. Conceptually, our results appear toreconcile the intra-cranial self-stimulation phenomenon withthe earliest view of rewards as ‘‘drive inhibitors’’ (Hull, 1943).Consistently, activating the hypothalamic AgRP-positive ‘‘drive’’neurons counteract parabrachium-mediated satiety (Camposet al., 2016) while conveying negative valence (Betley et al.,2015). Interestingly, recent ﬁndings do show that stimulatingthe gut-brain axis with nutrients suppresses AgRP-neuronal ac-tivity (Beutler et al., 2017; Su et al., 2017), raising the possibilitythat vagal satiating/rewarding signals inhibit AgRP-eliciteddrives.Related to the above, we do note that separate groups of NGneurons have previously been associated with different alimen-tary functions (Altschuler et al., 1989; Shapiro and Miselis,1985), including chemosensory versus mechanosensorysignaling (Williams et al., 2016). These ﬁndings lead to thehypothesis that nodose neurons mediating reward (i.e., rightnodose neurons) may be particularly sensitive to nutritive signalswhereas those inducing satiation independently of reward (e.g.,left nodose neurons) may preferentially display responses tomechanical distention. This possibility is suggested by thestriking speciﬁcity with which the R-NG controls the effects ofFigure 6. Right Nodose-parabrachio-nigral Pathway Is Required for Vagal Effects on Food Intake(A–F) The polysynaptic pseudorabies PRV152-GFP was injected bilaterally into dorsal st riatum (A). Dense retrograde labeling was observed in right (B), but not left(C), nodose ganglion. In NTS, labeling observed in medial rostral (D) and caudal (E) areas restricted to gut terminal ﬁelds. In PBN (F), labeling restricted todorsolateral gut terminal ﬁelds.(G) Dopamine receptor antagonism DS abolished satiating effects of CCK; N = 8, two-way RM-ANOVA main effects of antagonist F[1,7] = 5.124, p = 0.058; CCKF[1,7] = 47.7, p 0.001; antagonist 3CCK F[1,7] = 13.3, p = 0.006. Post hoc t tests: CCK + striatal aCSF versus saline + striatal aCSF Bonferroni *p 0.001; CCK +striatal aCSF versus CCK + striatal antagonist Bonferroni p 0.08.(H) Left: Right, but not left, gut vagal deafferentation abolished satiating effects of CCK (group F[1,8] = 8.7, *p 0.02; injection 3group F[1,8] = 19.6, *p = 0.002).Right: Bilateral deafferentation produces similar effects to right deafferentation (two-way mixed ANOVA injection 3group F[1,8] = 18.4, *p = 0.003).(I) PBNdl /SNc lesions abolished the suppressive effects of CCK on food inta ke: N = 5 in each group, main effects of CCK F[1,8] = 33.1, p 0.001; lesion F[1,8] =0.89, p = 0.37; CCK 3lesion F[1,8] = 11.0, *p = 0.01.(J–L) Right, but not left, gut vagal deafferentation abolished Fos expression induced by CCK in PBNdl (J, left, left deafferentation, right, right deafferentation) andNTS (K, left, left deafferentation, right, right deafferentation). (L) Quantiﬁes Fos+cells. N = 5, one sample t test, PBN: t[8] = 6.183, Bonferroni *p 0.001; NTS: t[8] =8.202, Bonferroni *p 0.001. AP, area postrema; DMV, dorsal motor nucleus of the vagus; NTS, nucleus of the solitary tract; PBNdl, parabrachial nucleus,dorsolateral region; PBNel, parabrachial nucleus, externolateral part; PBNm, medial parabrachial nucleus. Data reported as mean ±SEM.See also Figure S6.Cell 175, 665–678, October 18, 2018 675 cholecystokinin on appetite. Consistently, we found that theR-NG is polysynaptically linked to dorsal striatum dopaminerelease. Overall, our data revealed a gut-originated ascendingpathway consisting of the right nodose, the parabrachio-nigralpathway, and its targets in dorsal striatum, where each nodewas found to be required for CCK actions on appetite. Divergentstimulus selectivity in left versus right NG may ultimately ratio-nalize such striking asymmetry in vagal central pathways.As mentioned, we identiﬁed a dorsolateral parabrachio-nigralpathway that is critical for the expression of reward behaviorsmediated by gut vagal afferents. Transsynaptic labeling of rightvagal origin targeted this pathway, which not only respondedto digestive intestinal hormones, but also induced reward behav-iors when stimulated. While consistent with previous studiesshowing that CCK may induce ﬂavor preferences (Pe´rez andSclafani, 1991), these ﬁndings contrast with the notion thatCCK engages CGRP-positive neurons of the externolateral para-brachial nucleus (Carter et al., 2013). While these authors do infact observe that CCK-induced activation of CGRP-positiveneurons likely derives from severe forms of satiety, their subse-quent studies also suggest that CGRP-positive neurons mayparticipate in more physiological forms of CCK-induced satiation(Campos et al., 2016). Therefore, future designs must moreprecisely explore parabrachial activation in response to guthormone administration under a variety of physiologicalconditions.Cervical vagal stimulation is an approved treatment for refrac-tory major depression (Carreno and Frazer, 2017). Interestingly,the stimulator is commonly implanted on the left side to avoidthe cardiac complications elicited by electrically exciting thesinoatrial node (Howland, 2014). Our results, on the otherhand, suggest that positive affective states may be more efﬁ-ciently induced by means of stimulating the right vagus nerve.Accordingly, one possible approach around cardiac complica-tions may be implanting the stimulator on vagal nerve segmentslocated at the vicinity of the upper gut. Future vagus nerve stim-ulation trials coupled to measurements of human striatal dopa-mine release may determine the extent to which upper gut vagalexcitation enhances dopamine signaling within reward pathwaysof depressed patients.STAR+METHODSDetailed methods are provided in the online version of this paperand include the following:dKEY RESOURCES TABLEdCONTACT FOR REAGENTS AND RESOURCE SHARINGdEXPERIMENTAL MODEL AND SUBJECT DETAILSBExperimental AnimalsdMETHOD DETAILSBPeripheral organs viral injectionsBNodose ganglia injectionsBStereotaxic viral injections and optical ﬁberimplantationBMouse strain C57/BL6JBMouse strain Ai14BMouse strain VGlut2-CreBMouse strain VGat-CreBAnatomical tracing studiesBMouse strain VGlut2-CreBMouse strain VGlut2-CreBMouse strain VGat-CreBRetrograde tracingBRetrograde tracing using Cre-dependent pseudo-typed rabies virus expressionBHistological proceduresBc-Fos measurementsBWhole-Mount Immunostaining and Gut Tissue ClearingBSurgical Procedure for Implantation of Gastric Cathe-ters and Microdialysis Guiding CannulaeBDopamine measurements during intra-gastricinfusionsBBrain infusions of dopamine antagonistsBElectromyogram electrodes, recordings and analysesBIn vivo vagus nerve trunk recordingsBIn vivo Electrophysiological recordingsBSlice electrophysiologyBBehavioral StudiesBFood intake and Fos induction upon CCK and LiClinjectionsdQUANTIFICATION AND STATISTICAL ANALYSISBAnalysis of Behavioral trialsBAnalysis of In-vivo electrophysiological dataBAnalysis of In-vivo nerve recordings and electromyo-gram datadDATA AND SOFTWARE AVAILABILITYSUPPLEMENTAL INFORMATIONSupplemental Information includes six ﬁgures and four videos and can befound with this article online at https://doi.org/10.1016/j.cell.2018.08.049.ACKNOWLEDGMENTSWe thank M. McDougle for assistance with behavioral assays, M. Klein and K.Buchannan for packaging the rabies constructs and vagal recordings, andmembers of R. Medzhitov lab (Yale) for assisting with the breathing and heartrate measurements. This work was supported by NIH, United States(R01CA180030 and R01DC014859 to I.E.d.A., R00DK094871 andR01DK116004 to G.d.L., 1R21DA040782-01A1 to X.-B.G., K01DK103832 toD.V.B., and T32DK007568 to M.M.K.).AUTHOR CONTRIBUTIONSI.E.d.A. conceived the study. I.E.d.A., W.H., L.A.T., S.J.S.-L., and G.d.L. de-signed experiments. G.d.L. developed viral approach to nodose. W.H., T.Q.,and G.d.L. performed gastrointestinal and vagal injections and histology.W.H., L.A.T., M.H.P., T.L.F., and J.F. performed stereotaxic surgeries, behav-ioral, electromyogram, and optogenetic experiments, and analyzed data. D.Q.performed satiation assays. W.H. and S.J.S.-L. performed tracing, histology,and imaging. D.V.B. and M.M.K. packaged rabies constructs. Z.-W.L. andX.-B.G. performed whole-cell patch-clamp experiments and analyzed data.L.A.T., W.H., M.H.P., and I.O.P. performed in vivo electrophysiology andanalyzed nerve recordings and single-unit data. I.E.d.A. wrote the manuscript.All authors interpreted all data and edited the manuscript.DECLARATION OF INTERESTSThe authors declare no competing interests.676 Cell 175, 665–678, October 18, 2018 Received: February 2, 2018Revised: July 8, 2018Accepted: August 16, 2018Published: September 20, 2018REFERENCESAltschuler, S.M., Bao, X.M., Bieger, D., Hopkins, D.A., and Miselis, R.R. (1989).Viscerotopic representation of the upper alimentary tract in the rat: sensoryganglia and nuclei of the solitary and spinal trigeminal tracts. J. Comp. Neurol.283, 248–268.Angya´n, L. (1975). Vagal inﬂuences on hypothalamic self-stimulation in the cat.Life Sci. 17, 289–292.Banﬁeld, B.W., Kaufman, J.D., Randall, J.A., and Pickard, G.E. (2003). 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Cell 153, 896–909.678 Cell 175, 665–678, October 18, 2018 STAR+METHODSKEY RESOURCES TABLEREAGENT or RESOURCE SOURCE IDENTIFIERAntibodiesGoat Anti-GFP antibody (FITC) Abcam Cat# ab6662 RRID: AB_305635Rabbit-anti-mCherry Abcam Cat# ab167453 RRID: AB_2571870Alexa Fluor 647 recombinant rabbit monoclonalanti-Neuronal Nuclei (NeuN)Abcam Cat# ab190565 RRID: AB_2732785Chicken polyclonal to Tyrosine HydroxylaseantibodyAbcam Cat# ab76442 RRID: AB_1524535Mouse anti-TH ImmunoStar Cat# 22941 RRID: AB_10731005Goat polyclonal to CGRP Abcam Cat# ab36001 RRID: AB_725807Alexa Fluor 488 conjugated afﬁnipure goatanti-chicken IgY (H L)Abcam Cat# ab150169 RRID: AB_2636803Anti-c-Fos Rabbit polyclonal to c-Fos Abcam Cat# ab 190289 RRID: AB_2231989TRITC-conjugated afﬁnipure Goatanti-Rabbit IgG (H+L)Jackson ImmunoResearch Labs Cat# 111-025-144 RRID: AB_2337932FITC-conjugated afﬁnipure goatanti-rabbit IgG (H+L)Jackson ImmunoResearch Labs Cat# 111-095-144 RRID: AB_2337978FITC-conjugated afﬁnipure DonkeyAnti-Goat IgG (H+L)Jackson ImmunoResearch Labs Cat# 705-095-147 RRID: AB_2340401TRITC-conjugated afﬁnipure DonkeyAnti-Goat IgG (H+L)Jackson ImmunoResearch Labs Cat# 705-025-147 RRID: AB_2340389Biotinylated Goat Anti-Rabbit IgG antibody Vector Laboratories Cat# BA-1000 RRID: AB_2313606Goat anti-Phaseolus Vulgaris Agglutinin(E+L), UnconjugatedVector Laboratories Cat# AS-2224 RRID: AB_10000080Rabbit anti-PHA-L Dako Cat# B275 RRID: AB_2315137Rabbit anti-FG Chemicon, Millipore Cat# AB153-I RRID: AB_2632408Chemicals, Peptides, and Recombinant ProteinsFluoroGold (FG) Manufacturer: Fluorochrome Inc;Purchased from Fisher scientiﬁcCat# NC0560981PHA-L Vector Cat# L-1110Red RetroBeads lumaﬂuor N/ACCK-SAP Advanced Targeting Systems Cat # IT-31SAP Advanced Targeting Systems Cat # IT-21CCK8 AnaSpec Cat # AS-20739Clozapine N-oxide (CNO) Enzo Life Sciences Cat # BML-NS105-0025Clozapine Sigma Cat # C6305-25MGcis-(Z)-Flupenthixol dihydrochloride Sigma Cat # F114Metroclopramide Hydrochloride Sigma Cat # M0763-10GCritical Commercial AssaysVECTASTAIN Elite ABC HRP Kit(Peroxidase, Standard)Vector Laboratories Cat# PK-6100Experimental Models: Organisms/StrainsMouse: C57BL/6J The Jackson Laboratory JAX: 000664Mouse: Slc32a1tm2(cre)Lowl/J The Jackson Laboratory JAX: 016962Mouse: Slc17a6tm2(cre)Lowl/J The Jackson Laboratory JAX: 016963Mouse: Slc6a3tm1.1(cre)Bkmn/J The Jackson Laboratory JAX: 006660Mouse: Agrptm1(cre)LowThe Jackson Laboratory JAX: 012899(Continued on next page)Cell 175, 665–678.e1–e11, October 18, 2018 e1 ContinuedREAGENT or RESOURCE SOURCE IDENTIFIERMouse: B6;129S-Gt(ROSA)26Sortm34.1(CAG-Syp/tdTomato)Hze/JThe Jackson Laboratory JAX: 012570B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J The Jackson Laboratory JAX: 007914Recombinant DNAAAVrg-pmSyn1-EBFP-Cre Dr. Zeng-Addgene AAVViral Service51507H129 NIH Center for Neuroanatomywith Neurotropic VirusDTK-TTSAD-DG-GFP Dr. Diego V. Boho´rquez,Assistant Professor DukeMedicine-GI and NeurobiologyN/AAAV5-EF1a-DIO-hChR2(H134R)-EYFP Dr. Karl Deisseroth-Universityof North Carolina’s Vector CoreN/AAAV5-EF1a-DIO-EYFP Dr. Karl Deisseroth-Universityof North Carolina’s Vector CoreN/AAAV-hSyn-DIO-rM3D(Gs)-mCherry Dr. Bryan Roth - University of NorthCarolina’s Vector CoreN/AAAV5-ﬂex-taCasp3-TEVp Dr. Nirao Shah - University of NorthCarolina’s Vector CoreN/AAAV5-CA-FLEX-RG Dr. Naoshige - UchidaUniversity ofNorth Carolina’s Vector CoreN/AAAV8.2-hEF1a-DIO-synaptophysin-EYFP Viral Gene Transfer Core, McGovernInstitute for Brain Research,Massachusetts Institute of TechnologyN/ACAV2-Cre-GFP Institut de Ge´ne´tique Mole´culairede Montpellier, France (Junyent andKremer, 2015)N/APRV-GFP NIH Center for Neuroanatomywith Neurotropic Virus152PRV-RFP NIH Center for Neuroanatomywith Neurotropic Virus614Software and AlgorithmsMATLAB R20 17b MathWorks https://www.mathworks.com/products/matlab/Ofﬂine Sorter Plexon http://plexon.com/products/ofﬂine-sorterPatchMaster 2.20 HEKA http://www.heka.com/index.htmlIgor Pro 6.36 WaveMetrics https://www.wavemetrics.com/EthoVision XT 11.5 Noldus https://www.noldus.com/animal-behavior-research/products/ethovision-xtLabView 2014 LabView http://www.ni.com/download/labview-development-system-2014/4735/en/GraphPad Prism 7 GraphPad https://www.graphpad.com/scientiﬁc-software/prism/Adobe design standard CS6 Adobe http://shop.adobe.com/store/adbehme/en_IE/pd/ThemeID.29250800/productID.249257000SPSS 21.0 IBM Predictive Software https://www.ibm.com/support/knowledgecenter/SSLVMBSpike2 version 7 CED (Cambridge Electronic Design Ltd) http://ced.co.uk/products/spkovinImageJ bundled with 64-bit Java1.8.0_112NIH ImageJ http://shop.adobe.com;https://imagej.nih.gov/ij/index.html(Continued on next page)e2 Cell 175, 665–678.e1–e11, October 18, 2018 CONTACT FOR REAGENTS AND RESOURCE SHARINGFurther information and requests for reagents should be directed to, and will be fulﬁlled by the Lead Contact, Ivan E. de Araujo (ivan.dearaujo@mssm.edu).EXPERIMENTAL MODEL AND SUBJECT DETAILSAll experiments presented in this study were conducted according to the animal research guidelines from NIH and were approved bythe Institutional Animal Care and Use Committee of The J.B. Pierce Laboratory.Experimental AnimalsA total of 247 adult male mice were used. Strain details and number of animals in each group are as follows:183 C57BL/6J (Jackson Laboratories stock #000664)9 VGat-ires-Cre (Slc32a1tm2(cre)Lowl/J (Jackson Laboratories stock #016962)40 VGlut2-ires-Cre (Slc17a6tm2(cre)Lowl/J (Jackson Laboratories stock #016963)2 Dat-ires-Cre (Slc6a3tm1.1(cre)Bkmn/J (Jackson Laboratories stock #006660)2 Dat-ires-Cre 3Ai14 = Dat-ires-Cre (Slc6a3tm1.1(cre)Bkmn/J (Jackson Laboratories stock #006660) 3Ai14 (B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J (Jackson Laboratories stock #007914)1 Agrp-ires-Cre 3Ai34D = Agrp-Cre (Agrptm1(cre)Low(Jackson Laboratories stock #012899)ContinuedREAGENT or RESOURCE SOURCE IDENTIFIEROtherIntraLipid 20%, emulsion Sigma I141-100MLTotally Light 2Go - non-caloric powderedﬂavor drink mix, cranberry and wild berryﬂavors; sugar-free, Splenda-sweetened4C Foods N/AImplantable Optical Fibers Doric Lenses, Canada MFC_200/240-0.22_6mm_ZF1.25(G)_FLTFormvar-Insulated Nichrome Wires(EMG recording)A-M system Cat # 762000Male Miniature Pin Connector Fits Model1800 / 3000 Headstage Leads (EMG recording)A-M system Cat # 520200Female Miniature Pin Connector Fits A-MSystems’ electrodes (EMG recording)A-M system Cat # 520100A-M Systems 1700 AC Ampliﬁer A-M systems Cat # 6900002mm, cut off 6kDa, Microdialysis Probe andguide cannulaCMA CMA-7,16 tungsten microwires, 35-mm diameter(Electrophysiological recordings)TDT systems Cat # OMN1005The minimally traumatic elastic cuff electrodes(Micro Cuff Sling (200 mm/3pol/2,5mm/ cableentry top)CorTec GmbH N/AInfra-red pulse oximeter Starr Life Sciences Corp MouseOx PlusCollar Clip Sensor MouseOx Plus Sensor,Starr Life Sciences CorpSLS-015 021MATLAB script for electromyogram detection de Araujo lab https://data.mendeley.com/datasets/hygyrwftg9/draft/ﬁles/b9568d83-3686-476a-bd1c-88d8b99bff91/EMGs.zip?dl=1MATLAB script for PCA calculation ofneuronal datade Araujo lab https://data.mendeley.com/datasets/hygyrwftg9/draft/ﬁles/cc3b5fd0-5343-4964-82f9-fc87003abca0/ePhys_Recordings.zip?dl=1Spike2 scripts to count neurogram andmyogram eventsde Araujo lab https://github.com/matthewperkins/WH_IdA_VagusPaperCell 175, 665–678.e1–e11, October 18, 2018 e3 3Ai34D (B6;129S-Gt(ROSA)26Sortm34.1(CAG-Syp/tdTomato)Hze/J (Jackson Laboratories stock #012570)10 Ai14 (B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J (Jackson Laboratories stock #007914)All mice used in experiments were individually housed under a 12 hr light/dark cycle. At the time of the experiments, animals were8–20 weeks old. Animals weighted approximately 25-28 g. All animals were used in scientiﬁc experiments for the ﬁrst time. This in-cludes no previous exposures to pharmacological substances or altered diets. Health status was normal for all animals.METHOD DETAILSThe following provides details on viral injections and ﬁber/array implantation for each mouse strain. In all cases, preoperative anal-gesia: 0.05mg/Kg Buprenorphine (s.c.); anesthetic: 2% Isoﬂurane throughout; postoperative analgesia: 5mg/Kg Carprofen (s.c.)twice per day for consecutive 3 days. All surgeries were processed having the animals placed on a heated pad (CMA 450; HarvardApparatus, Holliston, MA), and allowed to recover under infrared heat until they chose to reside in the unheated side of the cage.Peripheral organs viral injectionsThe viral constructs (CAV2-Cre-GFP or AAVrg-pmSyn1-EBFP-Cre) were loaded into a NanoﬁlTM36G beveled needle (World Preci-sion Instruments, Sarasota, FL) and SilﬂexTMtubing (World Precision Instruments, Sarasota, FL), connected to a NanoﬁlTM10 mlsyringe (World Precision Instruments, Sarasota, FL) and mounted on a Pump 11 Elite Nanomite (Harvard Apparatus, Holliston,MA). Upper gut: Multiple 0.03 ml injections were made at 0.01 ml/s into each submucosal puncture. Each animal received a total vol-ume of 1ml in the stomach, duodenum and proximal jejunum. Myocardium: a total volume of 1ml of viral construct was injected into theventricular muscle through the central tendon of diaphragm. Pulmonary lobe: a total volume of 1ml of viral construct was injected intothe right pulmonary lobe through central tendon of diaphragm. Trachea: a total volume of 0.5ml of viral construct was injected into thewall of the upper trachea between larynx and clavicle.Nodose ganglia injectionsThe vagus nerve was separated from the carotid artery with a Spinal Cord Hook (Fine Science Tools, Foster City, CA) until the nodoseganglion became accessible. Viral aliquots (AAV-hSyn-DIO-rM3D(Gs)-mCherry; AAV-EF1a-DIO-EYFP; AAV5-EF1a-DIO-hChR2(H134R)-EYFP; AAV5-ﬂex-taCasp3-TEVp; H129DTK-TT) or chemicals (CCK-SAP (250ng/ml; IT-31) or SAP (250ng/ml; IT-21))were loaded into a NanoﬁlTM36G beveled needle (World Precision Instruments, Sarasota, FL) and SilﬂexTMtubing (World PrecisionInstruments, Sarasota, FL). For each nodose ganglion, a total 0.5ml volume was delivered into two sites, rostral and caudal to thelaryngeal nerve branch, at 0.2ul/min using a NanoﬁlTM10 ml syringe (World Precision Instruments, Sarasota, FL) mounted on aPump 11 Elite Nanomite (Harvard Apparatus, Holliston, MA).Stereotaxic viral injections and optical ﬁber implantationThe following provides details on viral injections for each mouse strain. In all cases, injections were performed with a Hamilton 1.0mLNeuros Model 7001KH syringe, at a rate of 0.02mL/min. In what follows, and for each mouse strain, we ﬁrst list the viral constructinjected, then the relevant stereotaxic coordinates for the injections are described. When applicable, the relevant stereotaxic coor-dinates for optical ﬁbers implantation are described. Optical ﬁbers were obtained from Doric Lenses, Inc. (Canada) the outer diameterof which is 240 mm; core diameter is 200 mm; numerical aperture is 0.22. Stereotaxic coordinates are with respect to bregma, accord-ing to a standardized atlas of the mouse brain (Franklin and Paxinos, 2008).Mouse strain C57/BL6JInjection typeUpper gut-AAVrg-CreViral constructAAVrg-pmSyn1-EBFP-Cre (1 mL)These same animals were injected with Cre-dependent viral constructs in Nodose ganglia or dorsal motor nucleus of the vagus.Injection typeNodose gangliaViral constructAAV-hSyn-DIO-rM3D(Gs)-mCherry; AAV-EF1a-DIO-EYFP; AAV5-EF1a-DIO-hChR2(H134R)-EYFP; AAV5-ﬂex-taCasp3-TEVp (0.5 mL)Opto ﬁber CoordinatesNTS: AP:-7.5mm, ML: ±0.3mm, DV 5.0mm.AP: AP:-7.6mm, ML: ±0mm, DV 4.8mmInjection typedorsal motor nucleus of the vaguse4 Cell 175, 665–678.e1–e11, October 18, 2018 Viral constructAAV5-EF1a-DIO-hChR2(H134R)-EYFP (0.3 mL)Control viral constructAAV-EF1a-DIO-EYFP (0.3 mL)Viral injection CoordinatesAP:-7.5mm, ML: ±0.3mm, DV 5.5 -5.3mm.Opto ﬁber CoordinatesDMV: AP:-7.5mm, ML: ±0.3mm, DV 5.2mm.Mouse strain Ai14Injection typeSNc-AAVrg-CreViral constructAAVrg-pmSyn1-EBFP-CreAP:-3.6mm, ML: ±1.5mm, DV 4.0mm -4.2mm. 0.3mL each side.These same animals were injected with Cre-dependent Caspase in PBNdlViral constructAAV5-ﬂex-taCasp3-TEVpControl viral constructAAV-EF1a-DIO-EYFPAP:-5.3mm, ML: ±1.5mm, DV 4.0mm -4.2mm. 0.3mL each side.Mouse strain VGlut2-CreInjection typePBNdl-DIO-ChR2, opto ﬁbers in SNcViral constructAAV5-EF1a-DIO-hChR2(H134R)-EYFPAP:-5.3mm, ML: ±1.5mm, DV 4.0mm -4.2mm. 0.3mL each side.Opto ﬁber CoordinatesAP:-3.6mm, ML: ±1.5mm, DV 4.0mm.Injection typePBNel-DIO-ChR2, opto ﬁbers in CeL/CViral constructAAV5-EF1a-DIO-hChR2(H134R)-EYFP-WPRA-pAAP:-5.3mm, ML: ±1.6mm, DV 4.2mm -4.5mm. 0.3mL each side.Opto ﬁber CoordinatesAP:-1.6mm, ML: ±2.7mm, DV 4.5mm.Mouse strain VGat-CreInjection typePBNel-DIO-ChR2, opto ﬁbers in PBNelViral constructAAV5-EF1a-DIO-hChR2(H134R)-EYFP-WPRA-pAAP:-5.3mm, ML: ±1.6mm, DV 4.2mm -4.5mm. 0.3mL each side.Opto ﬁber CoordinatesAP:-5.3mm, ML: ±1.6mm, DV 4.0mm.Anatomical tracing studiesAnterograde tracing using Phaseolus vulgaris leucoagglutinin (PHA-L)PBN-PHA-L2.5% PHA-L in phosphate buffer (pH 7.4) was injected unilaterally by iontophoresis by passing a positive-pulsed (7 s on/off) current(4.5 mA for 13-20 min) through a glass micropipette of 15 mm of internal tip diameter. For the stereotaxic approach to the PBN, thecaudal pole of the inferior colliculus was used as anteroposterior landmark. Coordinates AP: 0.6 mm, ML: 1.3-1.5 mm, DV: 2.6 mmAnterograde tracing using Cre-dependent synaptophysin expressionAnatomical markers in all images shown are according to standardized atlases of the adult mouse brain. The following mouse strainsand injections were used for assessment of cell-speciﬁc anterograde and retrograde projections. Injections were performedunilaterally. The viral construct used was the Cre-dependent AAV8.2-hEF1a-DIO-synaptophysin-EYFP, which allows for analysisof Cre-inducible synaptophysin expression.Cell 175, 665–678.e1–e11, October 18, 2018 e5 Mouse strain VGlut2-CreInjection typePBNdl- synaptophysinInjection CoordinatesAP:-5.3mm, ML: ±1.5mm, DV 4.0mm – 4.2mm. 0.3mLMouse strain VGlut2-CreInjection typeRight nodose ganglia - synaptophysin 0.3mLMouse strain VGat-CreInjection typePBNel- synaptophysinInjection CoordinatesAP:-5.3mm, ML: ±1.6mm, DV 4.2mm – 4.5mm. 0.3mLRetrograde tracingCeL/C-FluoroGold1% FG 0.05mL was injected unilaterally, speed 0.05mL/20min. Coordinates AP:-1.6mm, ML: 2.7mm, DV 4.8mm.DS-FluoroGold2% FG in saline was injected unilaterally by iontophoresis by passing a positive-pulsed (7 s on/off) current (1.5 mA for 6 min) through aglass micropipette of 20-25 mm of internal tip diameter. Two injections 0.6 mm apart were made along the dorsoventral axis.Coordinates AP: 1 mm, ML: 1.4 mm, DV: 2.2 mm and 2.8 mm.SNc-RetroBeads0.3mL RetroBeads was injected unilaterally, speed 0.02mL/min. Coordinates AP:-3.6mm, ML: 1.6mm, DV: 5.2mm – 5.4mm.Retrograde tracing using Cre-dependent pseudo-typed rabies virus expressionMouse strains VGat-Cre, Dat-Cre, Dat-Cre 3tdTomato, VGlut2-CreFor starter cells in SNc (AP:-3.6mm, ML: ±1.6mm, DV: 5.2mm – 5.4mm) or PBNdl (AP:-5.3mm, ML: ±1.5mm, DV 4.0mm– 4.2mm), AAV5-CA-FLEX-RG was unilaterally injected at 0.3mL. Two weeks afterward SAD-DG-GFP 0.3mL was injected on thesame site. Seven days later, the tissue were collected and then analyzed.Histological proceduresMice were deeply anesthetized with a ketamine/xylazine mix (400 mg ketamine + 20 mg xylazine kg body weight1I.P.). All animalswere perfused with ﬁltered saline, followed by 4% paraformaldehyde. Following perfusion, brains were left in 4% paraformaldehydefor 24 hours and then moved to a 20% sucrose solution in 0.02 M potassium phosphatebuffer (KPBS, pH 7.4) for 2 days. Brains werethen frozen and cut into four series 40 mm sections with a sliding microtome equipped with a freezing stage. To identify ﬁber andelectrode locations, relevant sections were identiﬁed and mounted on slides. Sections were then photographed under bright ﬁeldand ﬂuorescence. For Herpes Simplex Viruses visualization, 3 or 4 or 5 days after viral injection, mice were perfused and brainscut at 40mm. The tdTomato signal was ampliﬁed with Rabbit-anti-mCherry (Abcam, ab167453, 1:500) followed by TRITC-conjugatedafﬁnipure Goat anti-Rabbit (IgG (H+L) 111-025-144, Jackson Immuno, 1:200). For Cre-induced rM3D(Gs)-mCherry and hChR2-EYFPvisualization, 4 weeks after viral injection, mice were perfused, nodose ganglia and brain were collected and cut at 40mm. ThemCherry signal was ampliﬁed with Rabbit-anti-mCherry (Abcam, ab167453, 1:500) followed by TRITC-conjugated afﬁnipure Goatanti-Rabbit (IgG (H+L) 111-025-144, Jackson Immuno, 1:200). The EYFP signal was ampliﬁed with Goat anti-GFP antibody(FITC), (ab6662, Abcam, 1:500). For synaptophysin visualization, 4 weeks after viral injection, mice were perfused and brains cutat 40mm. The GFP signal was ampliﬁed with Goat anti-GFP antibody (FITC), (ab6662, Abcam, 1:500). For verifying the extensionof caspase-induced lesions, slices were incubated with Alexa Fluor647 recombinant rabbit monoclonal anti-Neuronal Nuclei(NeuN, Abcam ab190565, 1:500) antibody. For Tyrosine Hydroxylase visualization via immunoﬂuorescence, slices were incubatedwith chicken polyclonal to Tyrosine Hydroxylase antibody (ab76442, Abcam, 1:500), followed by Alexa Fluor488 conjugatedafﬁnipure goat anti-chicken (IgY (H L), ab150169, Abcam, 1:200). For CGRP visualization, slices were incubated with Goat polyclonalto CGRP (ab36001, Abcam, 1:500), followed by FITC-conjugated afﬁnipure Donkey Anti-Goat (IgG (H+L) 705-095-147, JacksonImmuno, 1:200) or TRITC-conjugated afﬁnipure Donkey Anti-Goat (IgG (H+L) 705-025-147, Jackson Immuno, 1:200). For FluoroGoldand RetroBeads experiments, seven days after injections animals were perfused as above and brains sliced in 40mm sections. Forvisualization of rabies expression, seven days after the rabies injections, animals were perfused and expression was observed incoronal sections at 160 mm intervals. Visualized cells were overlaid on a mouse brain atlas template.In experiments using Phaseolus vulgaris leucoagglutinin or Phaseolus vulgaris leucoagglutinin in combination with FluoroGold,after a survival of 5-10 days the animals were perfused as above and the brains sectioned at 30 mm. The sections were processed witha rabbit anti-PHA-L (1:5000; Dako, Carpenteria, CA) or with a rabbit anti-FG (1:10000; Bioscience Research Reagents, Temecula, CA)e6 Cell 175, 665–678.e1–e11, October 18, 2018 by using the ABC technique (Vectastain, Elite ABC kit 1:400; Vector). The peroxidase reaction product was revealed with the glucoseoxidase procedure and the metal-free 3,30-diaminobenzidine tetrahydrochloride (DAB) as the cromogen. An osmium tetroxide treat-ment was used to enhance the visibility of the labeling. An adjacent series of sections was stained for thionin. For the simultaneousdetection of PHA-L-labeled ﬁbers and tyrosine hydroxylase (TH)-positive mesencephalic neurons, sections were ﬁrst processed forPHA-L immunohistochemistry by using the nickel intensiﬁcation method and then immunostained for TH (with a mouse anti-TH1:5000; ImmunoStar, Hudson, WI) by using the metal-free DAB as the chromogen. A similar procedure was adopted for the simul-taneous detection of PHA-L-labeled ﬁbers and FG-positive neurons in the nigral complex, but in this case, a goat anti-PHA-L (1:4000;Vector) and a rabbit anti-FG (1:10000; Chemicon) were used as primary antibodies.c-Fos measurementsFor determining the effects of chemogenetic, CCK or LiCl stimulation on brainstem neuronal activity, 90 minutes after the appropriatestimulation, mice were sacriﬁced and perfused as described before. To visualize Fos immunoreactivity, either the ABC/DAB procedureor immunoﬂuorescence was used. Brieﬂy,brain sections was incubated with Rabbit Anti-c-Fos antibody(Abcam (ab190289), 1:10000),followed with Biotinylated Goat Anti-Rabbit IgG Antibody (BA-1000, Vector Laboratories,1:200), then reacted with avidin-biotin-perox-idase complex (‘‘ABC’’ method, Vectastain Elite ABC kit, Vector Laboratories, 1:200). A nickel diaminobenzidine (Nickel-DAB) glucoseoxidase reaction was used to visualize Fos-like immunoreactive cells. To visualize Fos via immunoﬂuorescence, slices were incubatedwith Rabbit Anti-c-Fos antibody (Abcam (ab190289), 1:2000), followed by TRITC-conjugated afﬁnipure goat anti-rabbit (IgG (H+L)111-025-144, Jackson Immuno, 1:200) or FITC-conjugated afﬁnipure goat anti-rabbit (IgG (H+L) 111-095-144, Jackson Immuno,1:200). Fos expression was analyzed and quantiﬁed as follows: Coronal sections at 160 mm intervals were photographed at 10 3magniﬁcation and montaged with Adobe Photoshopto to preserve anatomical landmarks. Fos+ neurons were counted with ImageJon each slice and expressed as the cumulative sum of Fos+ neurons within the relevant regions for each animal.Whole-Mount Immunostaining and Gut Tissue ClearingThe animals were anesthetized and perfused with saline and 1%PFA. The upper gut, heart and lung were dissected, opened andcleaned in a 1%PFA solution. Then the attached connective tissues were removed. After 24h ﬁxation with 4% PFA at 4C, the tissueswere washed with PBS for 1hr, which was then repeated twice. Tissues were then dehydrated at room temperature in a series ofmethanol/ddH2O solutions (20%, 40%,60%,80%,100%,100%), 30 min for each concentration. The tissue was next imbeddedwith a mixture of dichloromethane (Sigma)/methanol (2 volumes/1 volume) for 3hr, and then with 100% dichloromethane for15 min, twice. The tissue was then transferred to a cover-glass bottomed chamber and then ﬁnally cleared with 100% dibenzyl-ether(Sigma) for 1 hr twice, and prepared for confocal imaging.Surgical Procedure for Implantation of Gastric Catheters and Microdialysis Guiding CannulaeOnce animals had been anesthetized, a midline incision was made into the abdomen. The stomach was exteriorized through themidline incision and a purse string suture was placed in its non-glandular region, into which the tip of MicroRenathane tubing (Brain-tree Scientiﬁc Inc., Braintree, MA) was inserted. The purse string was tightened around the tubing, which was then tunneled subcu-taneously to the dorsum via a small hole made into the abdominal muscle; a small incision to the dorsum between the shoulder plateswas then made to allow for catheter exteriorization. Incisions were sutured and thoroughly disinfected and the exterior end of thecatheter plugged. Immediately after the above procedure, the animal was placed on a stereotaxic apparatus (David Kopf) under con-stant ﬂow of 2% isoﬂurane anesthesia and a circular craniotomy was drilled at AP = 1.5 mm, ML = ±1.5 mm implantation of a guidecannulae [DV = 1.5 mm from brain surface] for posterior insertion of a microdialysis probe into the dorsal aspect of the striatum (ﬁnalprobe tip positions [DV = 3.5 mm from brain surface]).Dopamine measurements during intra-gastric infusionsPrevious to, during, and after intra-gastric infusions (rate: 30mL/min; total volume: 0.6mL), microdialysate samples from mildly food-deprived awake mice freely moving in their home cages were collected, separated and quantiﬁed by HPLC coupled to electro-chem-ical detection methods (‘‘HPLC-ECD’’). Brieﬂy, after recovery from surgery, a microdialysis probe (2mm CMA-7, cut off 6kDa, CMAMicrodialysis, Stockholm, Sweden) was inserted into the dorsal striatum through the guide cannula (the corresponding CMA-7model). After insertion, probes were connected to a syringe pump and perfused at 1.2ml/min with artiﬁcial CSF (Harvard Apparatus).After a 40 min washout period and a subsequent 30 min pre-intake baseline sampling, dialysate samples were collected every sixminutes and immediately manually injected into a HTEC-500 HPLC unit (Eicom, Japan). Analytes were then separated via an afﬁnitycolumn (PP-ODS, Eicom), and compounds subjected to redox reactions within an electro-chemical detection unit (amperometric DCmode, applied potential range from 0 to 2000 mV, 1mV steps). Resulting chromatograms were analyzed using the softwareEPC-300 (Eicom, Japan), and actual sample concentrations were computed based on peak areas obtained from a 0.5pg/ml dopa-mine standard solution (Sigma) and expressed as % changes with respect to the mean dopamine concentration associated with thebaseline (i.e., pre-infusions) sampling period. Microdialysis sessions involving optical stimulation were performed identically asabove, except that after collecting the baseline, laser source was turned on for 12min at intermittent ON/OFF intervals of 30 s.Locations of microdialysis probes were conﬁrmed histologically. All experiments were performed on animals clearly alert and movingnaturally in their home cages.Cell 175, 665–678.e1–e11, October 18, 2018 e7 Brain infusions of dopamine antagonistsCannulae for striatal infusions were obtained from Plastics One (Roanoke, VA). The D1/D2 dopamine receptor antagonist ﬂupenthixol(Sigma) was infused bilaterally into the dorsal striatum at corrdinates AP = 1.5 mm, ML = ±1.5 mm DV = 2.5 at 15 mg/0.5 mL/hemi-sphere. The drug was prepared in aCSF (used as vehicle control) 5 minutes previous to the 1h-long oral intake tests.Electromyogram electrodes, recordings and analysesFirst, two twisted Formvar-Insulated Nichrome Wires (Diameter: Bare 0.002 inch. A-M system) were covered with polyethylene tubing(PE20, 0.15’’ x 0.45,’’ Braintree scientiﬁc). The tips of the nichrome wires were bared and exposed. One bare wire tip was soldered toa Male Miniature Pin Connector (520200, A-M Systems). The other bare wire tip was inserted through a 30G needle, and the tip bentand used for the implants into the stomach. For implants, the needle was then used for insertion of the wire into the stomach via asmall hole made into the abdominal muscle, with the bare wire hooked into the stomach. A suture was used to ﬁx the wire in place. Inorder to verify the efﬁcacy of the recordings, at the end of each session animals were administered the gut motility-inducing drugmetroclopramide hydrochloride (10mg/kg i.p.). Recordings were performed using the electromyogram module of a multichannelacquisition processor (Tucker-Davis Technologies, 3052Hz sampling rate). The male pin connector was attached to the femaleconnector, which had been soldered to a recording headstage. Laser pulses timestamps were synchronized to the recordings viaexternal TTL pulses into the TDT system. EMG signals from the stomach were recorded simultaneously to vagal nerve recordingsin the same animals.In vivo vagus nerve trunk recordingsThe vagus nerve trunk was separated from the carotid artery with a Spinal Cord Hook. The minimally traumatic elastic cuff electrodes(Micro Cuff Sling, 200 mm/3pol/2,5mm/cable entry top, CorTec GmbH) were gently placed under the nerve. The cuff electrode cableends were soldered to a Male Miniature Pin Connector (520200, A-M Systems) and attached to the female connector, which had beensoldered to a recording headstage. The surgery area was protected with mineral oil. Laser pulses were delivered directly to the nervetrunk by an Opto Probe Tips (Doric Lenses) connected to an Opto Probe Holder (Doric), and guided by a Micropositioner (P-10, WaferProbe). Laser pulses timestamps were synchronized to the recordings via external TTL pulses into the TDT system.In vivo Electrophysiological recordingsMice were placed on the stereotaxic apparatus and one electrode array consisting of 16 tungsten microwires (35-mm diameter,OMN1005, TDT systems) was implanted onto dorsal striatum (AP:+1.5 ML:1.5 DV:-2.5). Locations of electrodes were conﬁrmed his-tologically. Recordings were performed using a multichannel acquisition processor (Tucker-Davis Technologies) concomitantly tooptogenetic stimulation of nodose ganglion with blue light or green light. The multichannel processor recorded the laser pulses time-stamps and neuronal activities simultaneously. Only single neurons with action potentials of signal-to-noise ratios 3:1 wereanalyzed. The action potentials were isolated online using voltage-time threshold windows and a three-principal component contourtemplates algorithm. Spikes were resorted using the Ofﬂine Sorter software (Plexon, Inc.).Slice electrophysiologyThe coronal slices containing the parabrachial area (PBN) were prepared from C57B6 mice. Brieﬂy, mice were anesthetized with iso-ﬂurane and then decapitated. Then brains were rapidly removed and immersed in cold (4C) and oxygenated high-sucrose solutioncontaining (mM): sucrose 220, KCl 2.5, NaH2PO4 1.23, NaHCO326, CaCl21, MgCl26 and glucose 10, pH 7.3 with NaOH). After beingtrimmed to a small tissue block containing the PBN, coronal slices (300 mm thick) were cut on a vibratome and maintained at roomtemperature (23-25C) in a holding chamber with artiﬁcial cerebrospinal ﬂuid (ACSF) (bubbled with 5% CO2and 95% O2) containing(in mM): NaCl 124, KCl 3, CaCl22, MgCl22, NaH2PO41.23, NaHCO326, glucose 10, pH 7.4 with NaOH for recovery and storage. Afterrecovery at room temperature for at least one hour, slices were transferred to a recording chamber constantly perfused with ACSF ata temperature of 33C and a perfusion rate of 2 ml/min for electrophysiological experiments. Whole-cell patch clamp recording wasperformed in YFP-positive neurons in the parabrachial area under both voltage and current clamp. Micropipettes (3-4 MU) weremade of borosilicate glass (World Precision Instruments) with a Sutter P-97 micropipette puller and back ﬁlled with a pipette solutioncontaining (mM): K-gluconate 135, MgCl22, HEPES 10, EGTA 1.1, Mg-ATP 2.5, Na2-GTP 0.3, and Na2-phosphocreatin 10, pH 7.3with KOH. Both input resistance and series resistance were monitored throughout the experiments and the former was partiallycompensated. Only recordings with stable series resistance and input resistance were accepted. To stimulate neurons with anoptogenetic method, an LED-generated blue light pulses at different frequencies (5, 10 and 20 Hz) were applied to recordedneurons. All data were sampled at 3-10 kHz, ﬁltered at 3 kHz and analyzed with an Apple Macintosh computer using Axograph X(AxoGraph).Behavioral StudiesBehavioral apparatus for ﬂavor conditioning and dry lick testsBehavioral experiments were conducted in either one of three identical mouse behavior chambers enclosed in a ventilated andsound-attenuating cubicle (Med Associates Inc., St. Albans, VT). Each chamber is equipped with two slots for sipper tubing place-ments, at symmetrical locations on one of the cage walls. All sippers are connected to a contact-based licking detection devicee8 Cell 175, 665–678.e1–e11, October 18, 2018 allowing for measurements of licking responses with 10ms resolution. All lick timestamps were saved in a computer ﬁle for posterioranalysis. Software controlled lasers and infusion pumps equipped with TTL input devices were connected to the behavioral cham-bers and programmed to automatically trigger laser or intra-gastric infusions in response to the detection of licks. MED-PC IV (MedAssociates) was used as the platform for programming all experiments.StimuliNutritive intra-gastric stimuli were fat emulsions (IntraLipid, Baxter Healthcare, Deerﬁeld, IL). The stock emulsion contains as maincomponents 30% soybean oil, 1.2% egg yolk phospholipids, 1.7% glycerin, and water. The caloric density of 30% Intralipidis3.0 Kcal/mL, with 2.7 Kcal/mL accounted for by soybean oil and 0.3 Kcal/mL by phospholipids + glycerin. The original 30% emulsionwas diluted into an emulsifying control solution (1.2% phospholipids + 1.7% glycerine, in water) in order to prepare the 5% and 20%dilutions. Intra-gastric LiCl was prepared at 2.5mM/Kg, prepared in physiological saline, which was used as control stimulus.Flavor-nutrient conditioningFor rewarding conditioning tests, mice were trained to produce licks to spouts containing non-caloric ﬂavored solutions in order toreceive one intra-gastric infusion of either 20% IntraLipid or 5% IntraLipid depending on the identity of the ﬂavor being ingested. Thechoice of concentrations was based on previous studies demonstrating their effects during reward tests. For avoidance conditioningtests, protocols were identical except that intra-gastric infusions of 2.5mM/Kg LiCl were paired to one of the ﬂavors, and saline to theremaining ﬂavor. In all cases, the exterior part of the gastric catheter was connected to a segment of MicroRenathane tubing securedto the tip of a 3mL standard syringe containing the solutions to be infused and mounted on the syringe pump. The syringe pump wasplaced near a small hole made on the superior part of the sound attenuating box in such a way that mice could move freely inside thebehavioral chambers. During the task, the detection of the ﬁrst 200 licks triggered an intra-gastric infusion of the solution; the slowinfusions lasted for 20 min at a rate of 30mL/min (total volume: 0.6mL). Licks detected during and after the infusions had no pro-grammed consequences (i.e., did not result in additional infusions). This experimental design was chosen to eliminate possible con-founds related to differences in intake. For IntraLipid tests, ‘‘conditioning sessions’’ lasted for 1 hour and were performed for6 consecutive days under food (16h) deprivation, alternating daily the ﬂavor-lipid association. Thus, there were 3 sessions associatedwith each speciﬁc Flavor-lipid pair. For LiCl tests, ‘‘conditioning sessions’’ also lasted for 1 hour but were performed for only 2 consec-utive days and the rate of the infusion was adjusted to match the corresponding LiCl dose. Importantly, for each animal, ﬂavors werearbitrarily paired with either infusate, thereby preventing ﬂavor identity to inﬂuence preference formation. Short-term (5 min) two-bot-tle preference tests between the two distinct ﬂavors were performed previous to and following the conditioning sessions. These testswere performed in extinction, i.e., in the absence of intra-gastric infusions. Short-duration of this test aims to minimize postingestiveinﬂuences. After conditioning, an identical test was performed to assess the formation of ﬂavor preferences. The number of licks foreach Flavor was recorded and used to calculate the preference ratio as follows:Preference ratio for Flavor1=nðFlavorx1ÞnðFlavorx1Þ+nðFlavorx2Þwhere n(Flavorx) denotes the detected number of licks to Flavor xduring a given session. To eliminate the inﬂuence of side-biasesmice were tested four consecutive times with sipper positions being switched between any two consecutive tests, with the overallaverage across trials deﬁned as the actual preference. Flavor conditioning sessions where optical stimulation was used as theunconditioned stimulus were performed identically as above, except that in this case licks triggered 9sec-long blue light pulses(473-nm) via TTL pulses, coupling consumption to laser activation. Licks detected while the laser was on had no programmedconsequences.Dry lick-triggered intra-gastric feedingMice were trained to produce licks to a dry metallic spout in order to receive intra-gastric infusions of the fat emulsions. The exteriorpart of the gastric catheter was connected to a segment of MicroRenathane tubing secured to the tip of a 3 mL standard syringe con-taining the solutions to be infused and mounted on the syringe pump. The syringe pump was placed near a small hole made on thesuperior part of the sound attenuating box in such a way that mice could move freely inside the behavioral chambers. During thetask, a detected dry lick triggered an intra-gastric infusion of the fat emulsion that lasted for 6 s at a rate of 0.6 mL/min. However, licksdetected while an infusion was taking place had no programmed consequences (i.e., did not result in additional infusions). Experi-mental tests lasted for 1 hour. Animals were tested once a day in their responses to one single concentration of IntraLipid (20%). Inorder to train the animals in this task, once mice had recovered from surgery and been habituated to the behavioral chambers, a smallamount of standard chow was placed behind the spout’s oriﬁce (so that they could be smelled but not reached) to prime naive animalsto dry lick and obtain intra-gastric infusions. Training sessions lasted for 1 h and were performed daily under food (20 h) deprivation.After 4 priming sessions, clean odorless spouts replaced the ones containing chow. Animals were considered trained to perform theexperiments once they showed less than 10% between-session variability, a criterion reached within 10 consecutive sessions.Online place preference and avoidance tests (optogenetics)Place preference tests made use of automated video analyses (EthoVision XT11.5, Noldus). A rat behavioral cage was prepared insuch a way that the ﬂoor of one half of the cage was covered by bedding (preferred by all mice) and the ﬂoor of the other half of thecage was covered by an acrylic platform (less preferred by all mice). To demonstrate place preferences, the less preferred area of thetest cage was associated with switching the laser source ON, in such a way that the presence of the mouse in this area of the cageCell 175, 665–678.e1–e11, October 18, 2018 e9 switched the laser source ON via a TTL board under control of the camera monitoring. To demonstrate place avoidance, the preferredarea of the test cage was associated with switching the laser source ON. Quantiﬁcation of occupancy of different areas of the cagemade use of automated video analyses (EthoVision XT11.5, Noldus).Acquired place preference (chemogenetics)A rat behavioral cage was prepared in such a way that the ﬂoor of one half of the cage was covered by bedding (preferred by all mice),whereas the ﬂoor of the other half of the cage was covered by an acrylic platform (less preferred by all mice). During conditioning,transit between the two halves was blocked. In alternate daily sessions, the less preferred area of the test cage was associatedwith CNO injections and the preferred area of the test cage associated with vehicle control injections. Before placing the animalsin the corresponding half, the exterior part of the gastric catheter was connected to a segment of MicroRenathane tubing securedto the tip of a 3mL standard syringe containing the solutions to be infused and mounted on the syringe pump. During all sessions,mice were administered with an intra-gastric infusion of 0.3kCal IntraLipid (rate: 30 mL/min; total volume: 0.6mL of 5% IntraLipid).Both previous to and after the 6 1h-daily conditioning sessions, a 10min preference test was performed in such a way that the animalwas free to transit between the two halves of the test cage. Quantiﬁcation of occupancy of different areas of the cage made use ofautomated video analyses (EthoVision XT11.5, Noldus).Nose poke task (self-stimulation behavior)Mice were placed in an operant box equipped with two slots for nose poke slots at symmetrical locations on one of the cage walls.Nose poke slots were connected to a photo-beam detection device allowing for measurements of responses with 10-ms resolution,and only one of them triggered the 1 s-long laser pulses. The active side was counterbalanced among animals, and responsesdetected while the laser was on had no programmed consequences. Three consecutive days of 30 min stimulation sessions wereperformed, and on the fourth day one 30min-long extinction test was performed (i.e., both nose poke detectors were inactive). Time-stamps associated with all behavioral and laser events were saved in a computer ﬁle for posterior analysis.Optical Stimulation regimensStimulation frequencies were chosen according to the outcome of the nerve or slice electrophysiological studies. Although we did notdetect major differences between stimulation frequencies in terms of evoking (or failing to) behavior, we used the 1Hz frequencies fornodose ganglia terminals stimulation, and for either VGlut2-positive or VGat-positive parabrachial neurons and/or terminals, a 10Hzfrequency was chosen. Intensity at tip of ﬁbers was estimated at approximately 5 mW.Chemogenetic activationClozapine-N-Oxide (1mg/kg, EnZo Life Sciences) or Clozapine (0.1mg/kg, Sigma) was injected i.p., 10mins before the start of thebehavioral sessions.Food intake and Fos induction upon CCK and LiCl injectionsMice were single caged and 2.5g chow/day food restricted and divided into three groups: Saline-injected, CCK-injected and LiCl-injected. The CCK (10 mg/kg, AnaSpec Inc. Fremont, CA) and LiCl (2.5mg/kg) doses were chosen for producing a statistically identical50% reduction in food intake. Fifteen minutes after the injections animals were allowed ad libitum access to chow and after 1hour freeconsumption, the pellets were removed and weighted. For Fos studies, mice were also single caged and 2.5g chow/day foodrestricted and divided into three groups: Saline-injected, CCK-injected and LiCl-injected. The same CCK and LiCl doses chosenfor producing a statistically identical 50% reduction in food intake were used (CCK 10 mg/kg, LiCl 2.5mg/kg), although for Fos mea-surements no food intake was allowed after the injections. Animals were perfused 90 minutes after the administration of theinjections.Oxygen consumption, respiration and heart rate measurementsBoth breathing and heart rates were measured by pulse oximetry using the MouseOx Plus (Starr Life Sciences Corp, USA) in accor-dance with manufacturer’s instructions. Brieﬂy, each mouse was mildly anaesthetized using 5% isoﬂurane and shaved in the neck toplace a Collar Clip Sensor. Measurements were performed under mild anesthesia to collect representative, error-free data due to themotion artifacts.Oxygen consumption was measured via indirect calorimetry using the Oxymax/CLAMS Animal Monitoring System (Columbus In-truments, Columbus, OH), a mouse-dedicated, 4-cage system equipped with open-circuit calorimetry. Metabolism-induced heatwas derived by assessing the exchange of oxygen for carbon dioxide that occurs during metabolic processes, as measured bythe mass ﬂow principle. Oxygen (O2) measurement was performed via paramagnetic sensing and carbon dioxide (CO2) by singlebeam non-dispersed IR. The respective volumes were determined as:VO2=ViO2i VoO2o (1)VCO2=VoCO2o ViCO2i (2)where: Vi= Mass of air at chamber input per unit time, Vo= Mass of air at chamber output per unit time, O2i= Oxygen fraction in Vi,CO2i= Oxygen fraction in Vi,O2o= Carbon Dioxide fraction in Vo,CO2o= Carbon Dioxide fraction in Vo.e10 Cell 175, 665–678.e1–e11, October 18, 2018 QUANTIFICATION AND STATISTICAL ANALYSISData analyses, excluding all electromyogram/electrophysiological data, were performed using SPSS (v.21.0, IBM Predictive Soft-ware), Ethovision XT 11.5 (Noldus), GraphPad Prism 7 (GraphPad) and MATLAB (v.17b, MathWorks), Spike2 (Cambridge ElectronicDesign Ltd). Animals assigned to the different experimental groups were experimentally naive littermates, so that no randomization orother a priori criteria were adopted for group assignments. Experimental manipulations were analyzed according to within-subjectrepeated-measures designs. Order of experimental conditions was randomly assigned across subjects. Samples sizes were chosenbased on our previous studies employing similar optogenetic, electrophysiological and neuronal ablation approaches. Samples sizesadopted in our current study were sufﬁcient for detecting strong effect sizes while complying with guidelines from local enforcingrules requesting minimal animal usage by J.B. Pierce’s Institutional Animal Care and Use Committee. Experimenters were not blindto experimental conditions. Only animals carrying signs of distress/infection/bleeding/anorexia after the surgical procedures wereexcluded. Data from all animals used in the experiments were included in the ﬁnal analyses and plots.Analysis of Behavioral trialsFor all behavioral studies, including those resulting from pathway-targeted lesions, optogenetics and/or chemogenetic experiments,analyses made use of standard linear models (Pearson correlation), as well as one- or two-way (repeated-measures) ANOVAs andpost hoc t tests tests whenever relevant, for correcting for multiple comparisons. All data were reported as mean ±SEM. In all casessample sizes (N) denote number of animals used. All p values associated with the t tests performed correspond to two-tailed tests,and all post hoc tests were corrected for multiple comparisons by employing Bonferroni correction. To assess potentially spuriousresults associated with non-normality, all signiﬁcant effects were conﬁrmed by rerunning the tests using the appropriate non-para-metric test. All data are individually plotted (Prism 7, GraphPad), and the corresponding bar plot of the precision measures (mean ±SEM) were overlaid on the ﬁgure. The exact value of all N (always number of animals), df, T/F, and p values are reported in the ﬁgurelegends. An effect was considered statistically signiﬁcant whenever the corresponding statistic was associated with a p value (Bon-ferroni-corrected when appropriate) strictly less than 0.05.Analysis of In-vivo electrophysiological dataData were then imported into MATLAB (v.17b, MathWorks) for analysis using the custom-written software. Ninety-three single neu-rons displaying action potentials of signal-to-noise ratios 3:1, were analyzed. Otherwise data were discarded. To calculate ﬁringrates, instantaneous ﬁring rates were smoothed using the MATLAB ﬁltering function with a 60 s moving average using 50 msbins. To test for signiﬁcance in ﬁring rate changes, we used an individual unit analysis. A Kruskal-Wallis test (p 0.05) was performedon each unit to determine whether the mean ﬁring rate during the optogenetic period of stimulation was signiﬁcantly different from thebaseline mean ﬁring rate, and based on this criterium units were classiﬁed as modulated or not modulated. The baseline was takenfrom the start of session until the ﬁrst optical stimulation pulse. For all neurons recorded, the PSTH were arranged in a matrix (neu-rons = rows and bins = columns). The rows of the matrix were normalized as z-scores and then plotted using the ﬁrst component ofthe principal component analysis (PCA) (MATLAB toolboxes and custom-written scripts) aligned to optogenetic blue light stimulation(start or end respectively). In all cases, the ﬁrst component had a higher explained variance.Analysis of In-vivo nerve recordings and electromyogram dataTo perform the analyses, the signals were full-wave rectiﬁed and root mean square (RMS)-converted within moving windows of25-ms duration. Baseline signals (obtained from those periods during which the laser source was off) were used to establish acut-off Coffvalue deﬁned by the overall session mean Msand associated standard deviation STDsof the signals asCoff =Ms+ð33STDsÞThis cut off value was then used to determine whether the signal (S) at a given time point was signiﬁcantly different from baseline (i.e.SRCoff). Each time point in which this inequality holds true were deﬁned as an ‘‘event.’’ These analyses were performed usingcustom software programmed in MATLAB (v.17b, MathWorks) and are available upon request.DATA AND SOFTWARE AVAILABILITYCustom software is available. The accession number for the MATLAB script for electromyogram detection reported in this paperis [mendeley]: https://data.mendeley.com/datasets/hygyrwftg9/draft/ﬁles/b9568d83-3686-476a-bd1c-88d8b99bff91/EMGs.zip?dl=1. The accession number for the MATLAB script for PCA calculation of neuronal data reported in this paper is [mendeley]:https://data.mendeley.com/datasets/hygyrwftg9/draft/ﬁles/cc3b5fd0-5343-4964-82f9-fc87003abca0/ePhys_Recordings.zip?dl=1.The accession number for Spike2 scripts to count neurogram and myogram events reported in this paper is [github]: https://github.com/matthewperkins/WH_IdA_VagusPaper.Cell 175, 665–678.e1–e11, October 18, 2018 e11 Supplemental FiguresFigure S1. Characterization of the Effects Produced by Optogenetically Activating Nodose Neurons, Related to Figure 1(A–E) Confocal imaging of clariﬁed gastrointestinal, cardiac and lung tissue. The stomach and duodenum of wild-type mice were transfe cted with the retrogradelytransported construct AAVrg-pmSyn1-EBFP-Cre. The right nodose ganglion (R-NG) was then injected with the Cre-inducible Channelrhodopsin2 (DIO-ChR2-EYFP). A. Retrogradely transported AAVrg-pmSyn1-EBFP-Cre was readily detected in enteric neurons of the upper gastrointes tinal tract (Ai). In addition, AAVrg-pmSyn1-EBFP-Cre was detected in both R-NG (Aii) and left nodose ganglion (L-NG, Aiii). Bar plots (Avi) show that AAVrg-pmSyn1-EBFP-Cre transfectedapproximately the same number of cells in both R-NG and L-NG (N = 5 mice, paired t test t[4] = 1.4, p = 0.25), Bar = 100 mM. B-E. B. DIO-ChR2-EYFP expression inR-NG after retrograde injections into gut was restricted to the outermost neurons (magniﬁcation = 10 3, Bar = 100 mM). (C). DIO-ChR2-EYFP ﬁbers (green) werereadily detected throughout the upper, but not lower, gut segments. Bar = 100 mM. No ﬁber expression was detected in the heart (D) or lunge (E) after DIO-ChR2-EYFP was injected into R-NG. Bar = 100 mM.(F) Left, upper panel: Representative electromyogram trace of stomach musculature upon light stimulation of R-NG DIO-ChR2-EYFP-positive neurons. The traceshows thatlight pulses elicited no gut motilityresponses (laserpulses marked as blue bars on top).Left, lower panel: As positive control forthe gastric electromyogramrecordings, metroclopramide hydrochloride injectionswere found to produce robust enhancements of electromyogram activity. Right panel: Quantiﬁcation of supra-threshold events during stomach electromyogram recordings performed under optogenetic stimulation. n = 12; paired t test t[11] = 0.5 p = 0.6.(G) Fos expression in NTS and AP after optical activation of R-NG or L-NG neurons. Bar = 100 mM. In light control animals not carrying ChR2 in NG and with opticalﬁbers implanted on NTS (CTL-NG/NTS) or AP (CTL-NG/AP), no Fos was detected on either NTS or AP (Gi and Giv). In animals carrying ChR2 in L-NG, robust(legend continued on next page) Fos expression was observed in mice with optical ﬁbers implant ed on AP (L-NG/AP, Gii) but not in mice with optical ﬁbers implanted on NTS (L-NG/NTS, Gv).In animals carrying ChR2 in R-NG, robust Fos expression was observed in mice with optical ﬁbers implanted on NTS (R-NG/NTS, Giii) but not in mice withoptical ﬁbers implanted on AP (R-NG/AP, Gvi). Quantiﬁcation of Fos expression patterns reveal signiﬁcantly greater Fos activation in NT Sin R-NG/NTS micecompared to the other groups (one-way between-groups ANOVA F[2,12] = 156.5, *p 0.0001; pairwise comparisons R-NG/NTS versus other groups Bon-ferroni p 0.05). Similarly, analyses reveal signiﬁcantly greater Fos activation in AP in L-NG/AP mice compared to the other groups (one-way between-groupsANOVA F[2,12] = 37.6, *p 0.001; pairwise comparisons L-NG/AP versus other groups Bonferroni p 0.05).(H) Intake, for each ﬂavor, during ﬂavor conditioning upon blue laser stimulation of R-NG/NTS. Main effect of laser F[1,5] = 3.2,p = 0.13, Main effect of con-ditioning session F[2,10] = 1.8,p = 0.21; laser 3session interaction effect F[2,10] = 2.531,p = 0.128. Thus, in the absence of calories, laser pulses do not affectintake despite inducing preferences.(I) Optical stimulation of R-NG/NTS induced robust satiety during 20% IntraLipid intake tests. Shaded blue area indicates sessions when laser was ON for theON condition. N = 6, two-way RM-ANOVA, laser 3session interaction: F[8,40] = 19.8, *p 0.001.(J–R) Control experiments: Blue laser activation in the absence of nodose injections of Channelrhodopsin2 constructs (J). K. Left panel: Representa tive trace fromvagal nerve recording upon light stimulation of R-NG DIO-ChR2 -EYFP-negative neurons. The trace shows that light pulses elicited no nerve responses (laserpulses marked as blue bars on top). Right panel: Quantiﬁcation of supra-threshold events during vagus nerve recordings. n = 9; paired t test t[8] = 1.5 p = 0.16. L.Left panel: Representative electromyogram trace of stomach musculature upon light stimulation of R-NG DIO-ChR2-EYFP-negative neurons. The trace showsthat light pulses elicited no gut motility responses (laser pulses marked as blue bars on top). Right panel: Quantiﬁcatio n of supra-threshold events during stomachelectromyogram recordings. n = 9; paired t test t[8] = 0.53 p = 0.60. M. In the absence of the DIO-ChR2-EYFP construct in R-NG, R-NG(CTL)/NTS opticalstimulation fails to sustain self-stimulation behavior. N = 5; Two-way RM-ANOVA, Main effect of poking on the laser-pa ired hole versus inactive hole F[1,4] = 1.18,p = 0.33. Mice also failed to gradually increased the number of responses over daily sessions: Main effect of session, F[2,8] = 3.6,p = 0.07. Increases in responserates were not speciﬁc to poking on the laser-paired hole: Laser 3session interaction effect: F[2,8] = 0.04, p = 0.95. During laser-off extinction tests, mice failed topoke more on the laser-paired hole, paired t test t[4] = 0.72, p = 0.5. N. In the absence of the DIO-ChR2-EYFP construct in R-NG, R-NG(CTL)/NTS opticalstimulation failed to induce place preferences. The laser switch was on whenever the mouse was detected on the less preferred side of the cage. Left panel:Representative heatmap showing the pre-test baseline (upper) and on-line place preference (lower). Right panel: Place preference for laser-paired side, N = 5;paired t test t[4] = 2.2 p = 0.09. O. Left panel: In the absence of the DIO-ChR2-EYFP construct in R-NG, R-NG(CTL)/NTS optical stimulation failed to induce ﬂavorpreferences. Post-conditioning ﬂavor preferences for laser-paired ﬂavors N = 5; paired t test t[4] = 0.34, p = 0.74. For one-sample t tests against 50% (indif-ference) preferences: Pre-conditioning: t[4] = 0.29, p = 0.78; Post-conditioning, t[4] = 0.4, p = 0.7. Right panel: Intake for each ﬂavor during ﬂavor conditioning forblue laser in the absence of the DIO-ChR2-EYFP construct in R-NG. Main effect of laser F[1,4] = 2.3, p = 0.2, Main effect of conditioning session F[2,8] = 0.382,p =0.694; laser 3session interaction effect F[2,8] = 0.04,p = 0.96. P. Optical stimulation of R-NG(CTL)/NTS in the absence of the ChR2 construct failed to inducesatiation during 5% IntraLipid intake tests. Shaded blue area indicates sessions when laser was ON for the ON condition. N = 5, two-way mixed effects ANOVA,group 3session interaction: F[8,40] = 19.8, p 0.001. Q. Same as in Pbut for 20% IntraLipid; F[8,40] = 19.8, p 0.001. R. Optical stimulation of R-NG(CTL)/NTSin the absence of the DIO-ChR2-EYFP construct failed to alter chow intake. N = 5, three daily sessions (laser on day 2), one-way repeated-measures ANOVAF[2,8] = 2.1, p = 0.17).(S–V) Neuronal population dynamics in dorsal striatum during vagal afferent stimulation. Mice were injected with AAVrg -pmSyn1-EBFP-Cre in the upper gut, andwith DIO-ChR2-EYFP in the right nodose ganglion (R-NG). Optical ﬁbers were placed above the R-NG terminals in NTS. Additionally, a 16-wire array was im-planted in DS for electrophysiological striatal recordings. S: Dorsal striatal recordings in awake, freely moving mice during optogenetic stimulation of R-NGterminals in NTS. Left: The heatmap displays the ﬁrst principal component associated with the z-scores of all 112 individual neurons recorded, ranked accordingto the z-score values (low to high) during the stimulation period. Blue marks above the heatmap represent blue laser pulses. The dark blue ticks on the left indicatethose neurons signiﬁcantly modulated by laser. Neurons were deemed modulated (either excited or inhibited) by optogenetic stimulation according to event-related statistical analyses of the Z-scores (Kruskal-Wallis rank test, p 0.001). The graph corresponds to an entire experimental session: 30min baseline, 30minoptical stimulation, and 30min post-stimulation, periods. Right: Neural activity of two representative neurons modulated by optogenetic stimulation. Data fromentire session is shown, with blue-shaded areas representing the laser on period. Blue laser ChR2 activation yielded signiﬁcantly greater numbers of modulatedcells compared to non-ChR2 blue laser controls (Fisher’s exact test p 0.001). T. Electrophysiological recordings in anesthetized mice. In this case vagal ﬁberactivity was monitored concomitantly to array neuronal recordings in dorsal striatum. Heatmap corresponding to the ﬁrst principal component of 42 striatal cellsrecorded during direct optical stimulation of right vagal trunk. Again, neurons were ranked according to the z-score values during the stimulation period. The darkcontinuous trace on top corresponds to optically evoked responses in the right vagus nerve. Blue ticks represent blue laser pulses. In control animals, blue laseractivation in the absence of ChR2 yielded only one non-modulated cell (out of 98, 1%). This is signiﬁcantly less than the number of modulated cells in thepresence of ChR2 (20%, Fisher’s exact test p 0.001). U. Post-stimulus time histograms of nine representative units in dorsal striatum modulated by opto-genetic R-NG stimulation in anesthetized mice. V. Explained variances for the principal component analyses associat ed with heatmaps shown in panels Sand(awake ChR2+) T(anesthetized ChR2+) above, and in ChR2-negative light control.(W–Z) Confocal imaging of clariﬁed gastrointestinal, cardiac and lung tissue. The stomach and duodenum of wild-type mice were transfected with the retro-gradely transported construct AAVrg-pmSyn1-EBFP-Cre. The left nodose ganglion (L-NG) was then injected with the Cre-inducible Channelrhodopsin2 (DIO-ChR2-EYFP). W. DIO-ChR2-EYFP expression in L-NG after retrograde injections into gut was restricted to the outermost neurons (magniﬁcation = 10 3, Bar =100 mM). X. DIO-ChR2-EYFP ﬁbers (green) were readily detected throughout the upper, but not lower, gut segments. Bar = 100 mM. No ﬁber expression wasdetected in the heart (Y) or lunge (Z) after DIO-ChR2-EYFP was injected into L-NG. Bar = 100 mM.(AA) Left, upper panel: Representative electromyogram trace of stomach musculature upon light stimulation of L-NG DIO-ChR2-EYFP-positive neurons. Thetrace shows that light pulses elicited no gut motility responses (laser pulses marked as blue bars on top). Right panel: Quantiﬁcation of supra-threshold eventsduring stomach electromyogram recordings performed under optogenetic stimulation. n = 9; paired t test t[8] = 0.53, p = 0.6. BB-HH. Control experiments.Optical ﬁbers were implanted above the NTS (in the area more intensely innervated by R-NG terminals) in mice injected with ChR2 into the L-NG.(BB) L-NG/NTS fails to sustain self-stimulation behavior. N = 5; Two-way RM-ANOVA, Main effect of poking on the laser-paired hole versus inactive hole F[1,4] =3.5, p = 0.13. Mice varied number of responses over daily sessions: Main effect of session, F[2,8] = 22.0, p = 0.001. However, these changes in response rateswere not speciﬁc to poking on the laser-paired hole: Laser 3session interaction effect: F[2,8] = 0.5, p = 0.6. No effects were observed during laser-off extinctiontests, paired t test t[4] = 0.87, p = 0.4.(CC) L-NG/NTS optical stimulation failed to induce place preferences. The laser switch was on whenever the mouse was detected on the less preferred side ofthe cage. Left panel: Representative heatmap showing the pre-test baseline (upper) and on-line place preference (lower). Right panel: Place preference for laser-paired side, N = 5; paired t test t[4] = 2.3 p = 0.075.(legend continued on next page) (DD) L-NG/NTS optical stimulation failed to induce place aversion. The laser switch was on whenever the mouse was detected on the more preferred side of thecage. Left panel: Representative heatmap showing the pre-test baseline (upper) and on-line place preference (lower). Right panel: Place preference for laser-paired side, N = 5; paired t test t[4] = 0.85, p = 0.4.(EE) L-NG/NTS optical stimulation failed to induce ﬂavor preferences. Post-c onditioning ﬂavor preferences for laser-paired ﬂavors N = 5; paired t test t[4] = 0.28,p = 0.78. For one-sample t tests against 50% (indifference) preferences: Pre-conditioning: t[4] = 1.1, p = 0.33; Post-conditioning, t[4] = 0.86, p = 0.43.(FF) Intake for each ﬂavor during ﬂavor conditioning for L-NG/NTS optical stimulation. Main effect of laser F[1,4] = 0.53, p = 0.5, Main effect of conditioningsession F[2,8] = 0.056, p = 0.94; laser 3session interaction effect F[2,8] = 0.87, p = 0.45.(GG) Optical stimulation of L-NG/AP, but not L-NG/NTS, induced robust satiety during 20% IntraLipid intake tests. Shaded blue area indicates sessions whenlaser was ON. N = 5, two-way mixed effects ANOVA, group 3session interaction: F[8,64] = 14.8, *p 0.001.(HH) Optical stimulation of L-NG/NTS failed to alter chow intake. N = 5, three daily sessions (laser on day 2), one-way repeated-measures ANOVA F[2,8] = 1.1,p = 0.3).(II–LL) Control experiments. Optical ﬁbers were implanted above the AP (in the area more densely innervated by L-NG terminals) in mice injected with ChR2 intothe R-NG. II. R-NG/AP optical stimulation failed to induce place preferences. The laser switch was on whenever the mouse was detected on the less preferredside of the cage. Left panel: Representative heatmap showing the pre-test baseline (upper) and on-line place preference (lower). Right panel: Place preference forlaser-paired side, N = 5; paired t test t[4] = 0.1, p = 0.8. JJ. R-NG/AP optical stimulation failed to induce place aversion. The laser switch was on whenever themouse was detected on the more preferred side of the cage. Left panel: Representative heatmap showing the pre-test baseline (upper) and on-line placepreference (lower). Right panel: Place preference for laser-paired side, N = 5; paired t test t[4] = 0.07, p = 0.94. KK. Optical stimulation of R-NG/AP failed to alterchow intake. N = 5, three daily sessions (laser on day 2), one-way repeated-measures ANOVA F[2,8] = 2.4, p = 0.14). LL. R-NG/NTS and L-NG/AP animals hadtheir dopamine levels measured in the absence of any optical stimulation. In the absence of stimulation dopamine levels drop slightly below baseline, at equalrates between the two groups, two-way mixed effects ANOVA group 3time interaction effect F[15,120] = 1.0, p = 0.44.(MM) The stomach and duodenum of wild-type mice were transfected with the retrogradely transported construct AAVrg-pmSyn1-EBFP-Cre. Wh en compared tonon-injected mice, no alterations were observed in either baseline food intake (after intraperitoneal saline injections) or after CCK injections, two-way mixedeffects ANOVA, N = 5, CCK effects F[1,8] = 83.8,###p 0.001; group effect F[1,8] 0.1, p 0.99; group 3time interaction effect F[1,8] = 0.002, p = 0.97. Post hoctwo-sample t tests between injected versus non-injected mice,#saline t[8] = 0.013, p = 0.99;##CCK t[8] = 0.006, p = 0.995. Data reported as mean ±SEM. Figure S2. Characterization of Vagal Afferent Terminal Fields, Related to Figure 2(A–D) The retrograde Cre-carrying construct AAVrg-pmSyn1-EBFP-Cre was injected into the upper gut, the heart, the trachea or the lung. In all cases, the Cre-inducible construct DIO-ChR2-EYFP was bilaterally injected into the nodose ganglia. A. Gut terminal ﬁelds occupied a distinctive area in ventromedial NTS frommore caudal to more rostral levels (i-iv). B. Heart terminal ﬁelds occupied a distinctive area in dorsomedial/dorsolateral NTS from more caudal to more rostrallevels (i-iv). C. Trachea terminal ﬁelds occupied a distinctive area in rostrolateral NTS (i-iv). D. Lung terminal ﬁelds occupied a distinctive area in caudodorsolateralNTS (i-iv).(E) The stomach and duodenum of wild-type mice were transfected with the retrogradely transported construct AAVrg-pmSyn1-EBFP-Cre. When compared tonon-injected mice, no alterations in oxygen consumption were observed Two-sample t tests between injected versus non-injected mice, N = 4 each group, t[6] =0.3, p = 0.7.(F) Optical activation of nodose ﬁbers in NTS does not alter oxygen consumption, N = 6, one sample t test laser ON versus OFF t[5] = 1.2, p = 0.27.(G) Neither R-NG/NTS nor L-NG/NTS optical stimulation altered heart rate levels: N = 5 each group; Main effect of R-NG versus L-NG group, F[1,8] = 0.009,p =0.99; Sampling time effect F[14,112] = 1.04,p = 0.41; group 3sampling time effect F[14,112] = 0.77,p = 0.69. Blue area represents period during which blue lasersource was ON.(legend continued on next page) (H) Neither R-NG/NTS nor L-NG/NTS optical stimulation altered breath rate levels: N = 5 each group; Main effect of R-NG versus L-NG group, F[1,8] =0.023,p = 0.88; Sampling time effect F[14,112] = 0.44,p = 0.95; group 3sampling time effect F[14,112] = 0.39, p = 0.97. Blue area represents period during whichblue laser source was ON.(I) AAVrg-pmSyn1-EBFP-Cre was injected into upper gut, and DIO-ChR2-EYFP into the dorsal motor (efferent) nucleus of the vagus (DMV).(J) Concomitant vagal trunk recordings (upper traces) and gastric electromyograms (lower traces) show that optical activation of upper gut-innervatin g DMVneurons resulted in stomach contractions. Right panel Quantiﬁcation of supra-threshold electromyogram events n = 5; paired t test laser on versus off t[4] = 17.7,*p 0.001.(K) Optical activation of upper gut-innervating DMV neurons failed to induce online place preferences (laser paired to less preferred location, N = 5, t[4] = 0.16,p = 0.8).(L) Optical stimulation of upper gut-innervating DMV neurons failed to induce dopamine release above baselines in dorsal striatum. Note that although laseractivation did increase dopamine levels (N = 5, two-way repeated-measures ANOVA laser switch 3time interaction effect F[15,60] = 3.2, *p = 0.001), this wasinsufﬁcient to elevate levels above baseline (comparisons against baseline levels F[15,60] = 1.7, p = 0.06).(M) Optical stimulation of upper gut-innervating DMV neurons failed to reduce chow intake. N = 5, three daily sessions (laser on day 2), one-way repeated-measures ANOVA F[2,8] = 1.8, p = 0.2.(N) Quantiﬁcation of Fos responses in NTS upon injections of CNO+I.G. 0.3kcal Intralipid; CNO alone; I.G. 0.3kcal Intralipid alone. N = 5 in each group; One-wayANOVA F[2,12] = 44.2, *p 0.0001.(O) Expression patterns of Fos responses in NTS (left) upon injections of CNO+I.G. 0.3kcal Intralipid (i); CNO alone (ii); I.G. 0.3kcal Intralipid alone (iii).Bar = 100 mM.(P) Quantiﬁcation of Fos responses in PBN upon injections of CNO+I.G. 0.3kcal Intralipid; CNO alone; I.G. 0.3kcal Intralipid alone. N = 5 in each group; One-wayANOVA F[2,12] = 97.3, *p 0.0001.(Q) Expression patterns of Fos responses in PBN (left) upon injections of CNO+I.G. 0.3kcal Intralipid (i); CNO alone (ii); I.G. 0.3kcal Intralipid alone (iii).Bar = 100 mM.(R) Intake for each ﬂavor during ﬂavor conditioning for CNO+5% IntraLipid I.G. versus Saline + 5% IntraLipid I.G. Main effect of CNO F[1,4] = 0.33,p = 0.6, Maineffect of conditioning session F[2,8] = 9.0,p = 0.09; CNO 3time interaction effect F[2,8] = 1.3,p = 0.324. Thus, in the absence of calories, CNO injections do notaffect intake although inducing preferences.(S) CNO injections robustly inhibited 5% IntraLipid intake during 1hr-sessions; N = 5; Two-way RM-ANOVA CNO 3sessions interaction effect F[8,32] = 12.9,*p 0.001.(T) CNO injections robustly inhibited 20% IntraLipid intake during 1hr-sessions; N = 5; Two-way RM-ANOVA CNO 3sessions interaction effect F[8,32] = 8.5,*p 0.001.(U) CNO injections signiﬁcantly enhanced dopamine release in dorsal striatum induced by 30% (1.8kcal, 0.6ml) I.G. IntraLipid, N = 6; Two-way RM-ANOVA CNO3time interaction effect F[18,72] = 6.7, *p 0.0001.(V) CNO injections produced attenuated enhancement of dopamine release in dorsal striatum when compared to release in combination with I.G IntraLipid: N = 6;Two-way RM-ANOVA CNO 3time interaction effect F[18,72] = 4.3, *p 0.001.(W) CNO injections in animals not injected with DREADD constructs failed to alter 5% IntraLipid intake during 1hr-sessions; N = 5; Two-way RM-ANOVA CNO 3sessions interaction effect F[8,32] = 0.6, p = 0.7.(X) CNO injections in animals not injected with DREADD constructs failed to alter 20% IntraLipid intake during 1hr-sessions; N = 5; Two-way RM-ANOV A CNO 3sessions interaction effect F[8,32] = 0.8, p = 0.6.(Y) CNO injections in animals not injected with DREADD constructs failed to induce place preferences. During conditioning, CNO injections were paired to the lesspreferred side of the cage. In all sessions animals were infused with 0.3kcal IntraLipid into the gut. Left panel: Representative heatmap showing the pre-testbaseline (upper) and CNO (lower) on the animal’s location. Right panel: Place preference for CNO-paired side, N = 5; paired t test t[4] = 0.13, p = 0.9.(Z) Left panel: Control group with CNO injections: Post-conditioning ﬂavor preferences for ﬂavors paired with 5% I.G. IntraLipid + CNO versus 5% I.G. IntraLipid +saline, N = 5; paired t test t[4] = 0.009, p = 0.993. For one-sample t tests against 50% (indifference) preferences: Pre-conditioning: t[4] = 2.6, p = 0.06; Post-conditioning, t[4] = 0.9, p = 0.37. Right panel: Intake for each ﬂavor during ﬂavor conditioning for CNO+5% IntraLipid I.G. versus Saline + 5% IntraLipid I.G. Maineffect of CNO F[1,4] = 1.7, p = 0.2, Main effect of conditioning session F[2,8] = 100.3, p 0.0001; CNO 3time interaction effect F[2,8] = 0.19, p = 0.68.(AA) Left panel: Control group with clozapine injections: Post-conditioning ﬂavor preferences for ﬂavors paired with 5% I.G. IntraLipid + clozapine versus 5% I.G.IntraLipid + saline, N = 5; paired t test t[4] = 0.02, p = 0.98. For one-sample t tests against 50% (indifference) preference s: Pre-conditioning: t[4] = 0.3, p = 0.97;Post-conditioning, t[4] = 0.01, p = 0.99. Right panel: Intake for each ﬂavor during ﬂavor conditioning for clozapine+5% IntraLipid I.G. versus Saline + 5% IntraLipidI.G. Main effect of clozapine F[1,4] = 0.03, p = 0.86, Main effect of conditioning session F[2,8] = 13.7, p = 0.003; clozapine 3time interaction effect F[2,8] = 0.35,p = 0.71. Data reported as mean ±SEM. Figure S3. Characterization of Transsynaptic Labeling of Central Vagal Pathways, Related to Figure 3(A) A Cre-dependent synaptophysin-EYFP construct was injected into the right nodose ganglion if VGlut2-ires-Cre mice. Similarly to nodose infection withtranssynaptic herpes contructs, synaptophysin expression is observed in the dorsal vagal complex (AP, DMV, NTS) from caudal to rostral (Ai-Aiv) levels.(B) Bi-Bix. VGlut2-ires-Cre mice were injected with the Cre-inducible, transsynaptic Herpes Simplex Viruses 1 strain H129DTK-TT into the right nodose ganglion(R-NG). Description of the expression patterns at 3, 4 and 5 days post-injection.(C) Retrobeads injections in SNc (Ci) result in strong retrograde labeling in PBNdl and PBNm, but no labeling in PBNel (Cii).(D) Di-Dvi. The viral construct AAV-ﬂex-taCasp3-TEVp, which induces Cre-dependent caspase expres sion, was bilaterally injected into the PBNdl of VGlut2-ires-Cre mice. After two weeks, H129DTK-TT was injected into the right nodose ganglion (R-NG). Description of the expression patterns at 4 days post-injection. Noteabsence of herpes labeling in PBNdl, VTA and SNc. Panel V shows the effects of control (left) and caspase (right) injections into PBN of VGlut2-ires-Cre mice asvisualized by NeuN staining.(E) Ei-Eiii. VGlut2-ires-Cre mice were injected with H129DTK-TT into the left nodose ganglion (L-NG). Description of the expression patterns at 5 days post-injection.(F) The duodenum of wild-type mice was transfected with the retrogradely transported construct AAVrg-pmSyn1-EBFP-Cre. The right nodose ganglion (R-NG)was then injected with H129DTK-TT. Description of the expression patterns at 5 days post-injection.(G) Gi-Gii. Selective vagal deafferentation of the gut previous to introducing the transsynaptic virus into R-NG. Description of the expression patterns at 5 dayspost-injection. Herpes infection was restricted to the DMV. 5N = motor trigeminal nucleus; 7N = motor facial nucleus; AP =area postrema;BnST = bed nucleus ofthe stria terminalis;CeA = central nucleus of the amygdala; CeM = central nucleus of the amygdala , medial division; DM = dorsomedial hypothalamic nucleus;DMV = dorsal motor nucleus of the vagus; DR = dorsal raphe nucleus; LC = locus coeruleus;LH = lateral hypothalamic area; LRt = lateral reticular nucleus; NTS =nucleus of the solitary tract; PAG = periaqueductal gray matter; PBN = parabrachial nucleus; PBNdl = parabrachial nucleus, dorsolateral part; PBNm = para-brachial nucleus, medial part; PBNel = parabrachial nucleus, externolateral part; PCRt = parvicellular reticular nucleus; PSTn = parasubtha lamic nucleus; PV =paraventricular thalamic nucleus; PVA = paraventricular thalamic nucleus, anterior part; PVH = paraventricular hypothalamic nucleus; SNc =Substantia nigra,pars compacta; RRF = retrorubral ﬁeld; VTA = ventral tegmental area. Bar = 100mM. Figure S4. Characterization of the Efferent Connections of the Lateral Parabrachial Region, Related to Figure 4(A) In PBNdl, within the region displaying Fos responses to CCK injections, no CGRP expression was observed.(B) No Fos expression in response to LiCl was observed in PBNdl, therefore this lateral parabrachial region neither responds to LiCl nor contains CGRP-positiveneurons.(C–E) Fos responses to CCK and LiCl in NTS. Unlike the lateral parabrachial region, the NTS does not show any conspicuous topographical organization inresponse to these two compounds.(legend continued on next page) (F) Quantiﬁcation of Fos expression patterns in response to CCK and LiCl in NTS. Main effect of treatment: N = 5 in each gro up, F[2,12] = 29.2, p 0.0001; Post hoctwo-sample t tests saline versus LiCl Bonferroni *p 0.001; saline versus CCK p = 0.08; LiCl versus CCK Bonferroni **p = 0.001.(G) FluoroGold deposit in dorsal striatum.(H) PHA-L deposit in PBNdl via iontophoresis. Insert image magniﬁes injection site, revealing that the spread of PHA-L (labeled cells) was contained to PBNdl.(I) The anterograde tracer PHA-L was iontophoretically injected into PBNdl, and the mesencephalon was stained for tyrosine hydroxylase expression. Doubleimmunohistochemistry reveals terminals of parabrachial origin (injection site shown in panel H) innervating dopaminergic cells of SNc. Double immunohisto-chemistry reveals terminals of parabrachial origin innervating dopaminergic cells of SNc.(J) PHA-L deposit in PBNel via iontophoresis. Insert image magniﬁes injection site, revealing that the spread of PHA-L (labeled cells) was contained to PBNel.(K) Darkﬁeld photomicrographs of coronal sections revealing a substantial anterograde labeling in the CeL/C region after the PHA-L injection into PBNel.(L and M) Fluorogold was injected in CeL/C, and retrograde labeling in the parabrachial area was restrict ed to PBNel.(N) Left panel: Strong Fos expression in CeL/C in response to LiCl injections. Note that Fos expression is robust and yet restricted to the site targeted by PBNel(compare against panel K). Right panel: Average Fos-positive cell counts per section in PBNel, N = 5 mice. Two sample t test *p 0.00001.(O and P) The SNc of both DAT-ires-Cre and VGat-ires-Cre mice were transfected of with the construct AAV5-EF1a-DIO-G, and two weeks afterward, the Cre-inducible retrograde pseudotyped rabies virus SADDG-GFP were transfected at the same site of the SNc. In both DAT-ires-Cre (O) and VGat-ires-Cre (P) mice, noevidence of rabies-infected cells was found on 7days post rabies infection in the dorsal vagal complex, including NTS.(Q) DAT-ires-Cre 3td-Tomato mice were unilaterally injected with AAV5-EF1a-DIO-G into SNc. Two weeks later SADDG-GFP was injected into the SNc of thesame animals. Seven days after SADDG-GFP injections, profuse overlap between Tomato+ and GFP+ cells was observed.(R) Robust SADDG-GFP expression was observed in both PBNdl and PBNm.(S and T) In control experiments in which DAT-ires-Cre 3td-Tomato mice were not injected with AAV5-EF1a-DIO-G, while SADDG-GFP expression is observedin the SNc injection site (S), no SADDG-GFP cells were observed in any parabrachial sector (T).(U and V) VGlut2-ires-Cre mice were unilaterally injected with AAV5-EF1a-DIO-G into PBNdl. Two weeks later SADDG-GFP was injected into the PBNdl of thesame animals (U). Seven days after SADDG-GFP injections, profuse expression of GFP+ cells was observed in ventromedial NTS, speciﬁcally where we hadobserved upper gut right nodose terminal ﬁelds (V).(W) In AgRP-ires-Cre;ﬂoxed-synaptophysin td-Tomato mice, AgRP-derived terminal ﬁelds were found to innervate the PBNdl, PBNel, and PBNm regions,including the parabrachial region containing CGRP+ neurons. 12N = hypoglossal nucleus; AP =area postrema;CeC = (Inter) Capsular subnucleus of the centralamygdalar nucleus; CeL = Lateral subnucleus of the central amygdalar nucleus; DS = Dorsal Striatum; DMV = dorsal motor nucleus of the vagus; NTS = nucleus ofthe solitary tract; PBN = parabrachial nucleus; SNc =Substantia nigra,pars compacta;VTA =ventral tegmental area. Bar = 100mM. Figure S5. Behavioral Characterization of Lateral Parabrachial Neuronal Populations, Related to Figure 5(A) Intake for each ﬂavor during ﬂavor conditioning for blue laser in PBNdl[VG lut2]/SNc. Main effect of laser F[1,5] = 3.2,p = 0.13, Main effect of conditioningsession F[2,10] = 5.4, p = 0.02; laser 3session interaction effect F[2,10] = 1.0, p = 0.37. Thus, in the absence of calories, laser pulses do not affect intake despiteinducing preferences.(B) PBNdl/SNc optical stimulation robustly decreased chow intake. N = 5, three daily sessions (laser on day 2), one-way repeated-measures ANOVA F[2,8] =106.9, *p 0.0001).(C) PBNdl/SNc optical stimulation induced robust satiety during 20% IntraLipid intake tests. Shaded blue area indicates sessions when laser was ON for the ONcondition. N = 5, two-way RM-ANOVA, laser 3session interaction: F[8,32] = 10.0, *p 0.001.(D) Cre-inducible Channelrhodopsin2 (DIO-ChR2-EYFP) was injected into the lateral parabrachial region of VGat-ires-Cre mice, and optical ﬁbers placed on thelateral parabrachial region.(E) Expression of the DIO-ChR2-EYFP construct was the clearest in PBNel.(F) Injections of Cre-inducible synaptophysin-EYFP into the PBNel of VGat-ires-Cre mice reveal a dense GABAergic termination ﬁeld in PBNdl. This suggests thatactivation of lateral parabrachial VGat neurons should induce avoidance behaviors, as they would in particular suppress PBNdl neurons projecting to SNc.Bar = 100mM.(G and H) After injections of Cre-inducible synaptophysin-EYFP into the PBNel of VGat-ires-Cre mice, no terminals in SNc were detected (G), whereas very weaklabeling was observed in CeC/CeL (H). Bar = 100mM.(legend continued on next page) (I) PBNel[VGat] optical stimulation induces place avoidance. Left panel: Representative heatmap showing the pre-test baseline (upper) and on-line placepreference (lower). Right panel: Place avoidance for laser-paired side, N = 5; paired t test t[4] = 7.4, *p = 0.002.(J) PBNel[VGat] optical stimulation during ingestion of 5% IntraLipid. After daily baseline sessions 1-3, intake is reduced during laser ON sessions 4-6, and returnto baseline levels was delayed on post-laser sessions 7-9. Dark green points represents laser OFF, and light green line represents laser ON, with blue areaindicating days in which laser was ON. N = 5, two-way RM-ANOVA laser 3session interaction effect F[8,32] = 9.5, *p 0.001.(K) Cre-inducible Channelrhodopsin2 (DIO-ChR2-EYFP) was injected into the PBNel of VGlut2-ires-Cre mice, and optical ﬁbers placed above parabrachialterminals on CeL/C (PBNel[VGlut2]/CeL/C pathway).(L) Injection of the DIO-ChR2-EYFP construct was restricted to PBNel.(M) Similar injections of Cre-inducible synaptophysin-EYFP into the PBNel of VGlut2-ires-Cre mice reveal dense glutamatergic parabrachial terminals in CeL/C.(N) PBNel[VGlut2]/CeL/C optical stimulation fails to sustain self-stimulation behavior. N = 6; Two-way RM-ANOVA, Main effect of poking on the laser-pairedhole versus inactive hole F[1,5] = 2.5,p = 0.17. Mice varied number of responses over daily sessions: Main effect of session, F[2,10] = 10.4, p = 0.004. However,these changes in response rates were not speciﬁc to poking on the laser-paired hole: Laser 3session interaction effect: F[2,10] = 0.8, p = 0.44. No effects wereobserved during laser-off extinction tests, paired t test t[5] = 2.16, p = 0.08.(O) PBNel[VGlut2]/CeL/C optical stimulation induces place avoidance. Left panel: Representative heatmap showing the pre-test baseline (upper) and on-lineplace preference (lower). Right panel: Place avoidance for laser-paired side, N = 6; paired t test t[5] = 12.3, *p = 0.001.(P) PBNel[VGlut2]/CeL/C optical stimulation induces moderate ﬂavor avoidance. Post-conditioning ﬂavor preferences for laser-paired ﬂavors N = 6; paired t testt[5] = 2.8, *p = 0.038. For one-sample t tests against 50% (indifference) preferences: Pre-conditioning: t[5] = 0.041,p = 0.969; Post-conditioning, t[5] = 1.889,p = 0.1.(Q) Intake for each ﬂavor during ﬂavor conditioning for blue laser in PBNel[VGlut2]/CeL/C. Main effect of laser F[1,5] = 11.6, p = 0.02, Main effect of conditioningsession F[2,10] = 2.0, p = 0.18; laser 3session interaction effect F[2,10] = 0.3, p = 0.7.(R) PBNel[VGlut2]/CeL/C optical stimulation during ingestion of 5% IntraLipid. After daily baseline sessions 1-3, intake is reduced during laser ON sessions 4-6,and immediately returned to baseline on post-laser sessions 7-9. N = 6, two-way RM-ANOVA laser 3session interaction effect F[8,40] = 8.9, *p 0.001. Theseresults show that circuits inducing reward versus avoidance behaviors may produce similar reduction s in intake. Data reported as mean ±SEM. Figure S6. Histological and Behavioral Characterization of Vagal and Parabrachio-nigral Lesions, Related to Figure 6(A–E) Retrogradely transported, polysynaptic pseudorabies viral constructs were injected into the right dorsal striatum (PRV152-GFP, A) and the left dorsalstriatum (PRV614-RFP, A). Bilateral, equally dense distribution of GFP+ and RFP+ cells were observed in VTA and SNc (B). Bilateral, equally dense distribution ofGFP+ and RFP+ cells were observed in PBN (C). Bilateral, equally dense distribution of GFP+ and RFP+ cells were observed in rostromedial NTS (D) and incaudomedial NTS (E). Bar = 100mM. These NTS regions correspond to the areas containing upp er gut nodose terminal ﬁelds. Note the rare GFP+ or RFP+ labelingwithin the dorsal motor nucleus of the vagus (DMV).(F) Infusions of the dopamine receptor antagonist into the dorsal striatum failed to disrupt the suppressing effects of lithium cholride (LiCl): antagonist 3LiClinteraction effect F[1,5] = 6.5, p 0.05. Post hoc t tests: Intraperitoneal LiCl + striatal aCSF versus intraperitoneal saline + striatal aCSF Bonferroni *p 0.006;Intraperitoneal LiCl + striatal aCSF versus intraperitoneal LiCl + striatal antagonist Bonferroni p 0.23.(G) Selective vagal deafferentation of the gut was achieved by injecting the neurotoxin saporin conjugated to cholecystokinin into the nodose ganglia.(H) Vagal deafferentation of the gut abolishes conditioned learning to ﬂavors paired to intra-gastric higher-calorie nutritive lipids versus ﬂavors paired to intra-gastric lower-calorie nutritive lipids; N = 6 in each group, Two-way mixed model ANOVA, main effect of intra-gastric infusate F[1,10] = 8.3, p = 0.016; main effect ofvagal gut deafferentation F[1,10] = 45.6, p 0.0001; Infusate 3deafferentation interaction effect F[1,10] = 41.6, *p 0.001.(I) Similar effects were observed after PBNdl/SNc lesions: N = 5 in each group, main effect of session F[9,72] = 3.1, p = 0.003; main effect of lesion F[1,8] = 26.4,p = 0.001; session 3lesion interaction effect F[9,72] = 4.7, *p 0.001.(J) Vagal deafferentation of the gut disrupts performance on a reward task where mice learn to lick a dry sipper to self-infuse nutritive lipids into the stomach.Deafferentated mice failed to gradually increase intra-gastric feeding over daily sessions: N = 5 in each group, Two-way mixed model ANOVA, main effect of(legend continued on next page) session F[9,72] = 1.9, p = 0.066; main effect of vagal gut deafferentation F[1,8] = 8.9, p = 0.017; session 3deafferentation interaction effect F[9,72] = 4.8,*p 0.001.(K) Vagal deafferentation of the gut fails to disrupt aversion learning to ﬂavors paired to intra-gastric malaise agents (LiCl) versus ﬂavors paired to intra-gastricvehicle; N = 5 in each group, Two-way mixed model ANOVA, main effect of intra-gastric infusa te F[1,8] = 89.6, p 0.0001; main effect of vagal gut deafferentationF[1,8] = 0.33, p = 0.58; Infusate 3deafferentation interaction effect F[1,8] = 0.94, p = 0.36.(L) Intake for each ﬂavor during the one-session ﬂavor avoidance learning after vagal deafferentation of the gut. Two-way mixed model ANOVA; Main effect ofdeafferentation F[1,8] = 0.11, p = 0.74, Main effect of treatment F[1,8] = 139.2, p 0.001; deafferentation 3treatment inter action effect F[1,8] = 0.34, p = 0.57.(M) Robust Fos induction in response to the malaise agent LiCl is observed in the PBNel even after gut vagal deafferentation. Left: Representative case of Foslabeling in PBNel of deafferentated mice in response to an intra-peritoneal injection of LiCl; Center: Representative case of Fo s labeling in PBNel of deafferentatedmice in response to an intra-peritoneal injection of saline. Right: Fos counts (average per section), two-sample t test LiCl versus saline, *p 0.01.(N) Vagal deafferentation of the gut abolishes dopamine release in dorsal striatum induced by intra-gastric infusions of nutritive lipids; N = 5 in each group , Two-way mixed model ANOVA, sampling time 3deafferentation interaction effect F[15,120] = 6.7, *p = 0 0.001.(O) Targeted ablation of the parabrachio-nigral pathway was achieved by ﬁrst transfecting the SNc of wild-type mice with the retrograde Cre-carrying constructAAVrg-pmSyn1-EBFP-Cre; next, PBNdl was transfected with the viral construct that induces Cre-dependent caspase expression.(P and Q) Targeted ablation of the parabrachio-nigral pathway via AAVrg-pmSyn1-EBFP-Cre injections in the SNc of TdTomato reporter mice, and Cre-dependent AAV-DIO-caspase3 expression in PBNdl. Control mice were injected with AAVrg-pmSyn1-EBFP-Cre in SNc and Cre-dependent AAV-GFP in PBNdl.Clear neuronal ablation in PBNdl can be observed in lesioned (Q), but not in control (P), mice. Note that the density and shape of the CGRP+ ﬁeld (green) in PBNelis comparable between the two sections.(R and S) In contrast, no neuronal loss could be detected in the pendunculopontine tegmentum (PPTg) of either control (R) or lesi oned (S) mice. PPTg is located inthe vicinity of PBN, and sends robust terminals to SNc.(T) Histological quantiﬁcation of the effects of lesioning the PBNdl/SNc pathway. Graphs show the number of Cre-positive cells per section in different PBN andPPTg regions. Only PBNdl was associated with signiﬁcant neuronal loss. N = 5, one-way ANOVAs PBNdl: F[1,8] = 54.704, Bonferro ni*p 0.001; PBNm: F[1,8]=0.63, p = 0.44; PBNel: F[1,8] = 0.27, p = 0.61; PPTg: F[1,8] = 0.32, p = 0.58; CGRP: F[1,8] = 0.432, p = 0.529.(U) Similar effects were observed after PBNdl- SNc lesions: N = 5 in each group, main effect of intra-gastric infusate F[1,8] = 47.8, p 0.001; main effect of lesionF[1,8] = 33.7, p 0.0001; Infusate 3lesion interaction effect F[1,8] = 32.8, *p 0.001.(V) Intake for each ﬂavor during ﬂavor conditioning after parabra chio-nigral lesions. Three-way mixed model ANOVA; Main effect of lesion F[1,8] = 1.37, p = 0.27,infusate 3conditioning session interaction effect F[2,16] = 0.88, p = 0.43; infusate 3conditioning session 3lesion interaction effect F[2,16] = 0.47, p = 0.62.Thus, despite deﬁcits in preferences, parabrachio-nigral lesions did not disrupt intake levels during conditioning.(W) Similar effects were observed after PBNdl/SNc lesions: N = 5 in each group, main effect of session F[9,72] = 3.1, p = 0.003; main effect of lesion F[1,8] = 26.4,p = 0.001; session 3lesion interaction effect F[9,72] = 4.7, *p 0.001.(X) Similar effects were observed after PBNdl/SNc lesions: N = 5 in each group, main effect of intra-gastric infusate F[1,8] = 114.9, p 0.001; main effect of lesionF[1,8] = 3.77, p = 0.088; Infusate 3lesion interaction effect F[1,8] = 1.0, p = 0.32.(Y) Intake for each ﬂavor during the one-session ﬂavor avoidance learning after parabrachio-nigral lesions. Two-way mixed model ANOVA; Main effect of lesionF[1,8] = 0.42, p = 0.53, Main effect of treatment F[1,8] = 103.6, p 0.001; lesion 3treatment interaction effect F[1,8] = 0.004, p = 0.95.(Z) Similar effects were observed after PBNdl/SNc lesions: N = 5 in each group, sampling time 3deafferentation interaction effect F[15,120] = 5.25, *p =0 0.001.(AA) The Cre-expressing retrograde construct AAVrg-pmSyn1-EBFP-Cre and nodose ganglia were injected with AAV-ﬂex-taCasp3-TEVp.(BB) Caspase induction signiﬁcantly reduced neuronal density in nodose ganglia. Bar = 100mM. N = 5,one-way ANOVA: F[1,8] = 148.1, p 0.001.(CC) Caspase-induced vagal deafferentation of the gut abolishes conditioned learning to ﬂavors paired to intra-gastric higher-calorie nutritive lipids versus ﬂavorspaired to intra-gastric lower-calorie nutritive lipids; N = 6 in each group, Two-way mixed model ANOVA, main effect of intra-gastric infusate F[1,10] = 20.3, p =0.001; main effect of vagal gut deafferentation F[1,10] = 72.4, p 0.0001; Infusate 3deafferentation interaction effect F[1,10] = 45.3, *p 0.001.(DD) Intake for each ﬂavor during ﬂavor conditioning after caspase-induced vagal deafferentation of the gut. Three-way mixed model ANOVA; Main effect ofdeafferentation F[1,10] = 0.14, p = 0.7, infusate 3conditioning session interaction effect F[2,2 0] = 0.12, p= 0.88; infusate 3conditioning session 3deafferentationinteraction effect F[2,20] = 0.01,p = 0.99. Thus, despite deﬁcits in preferences, gut vagal deafferentation did not disrupt intake levels during conditioning .(EE) Caspase-induced vagal deafferentation of the gut disrupts performance on a rewardtask where mice learnto lick a dry sipper to self-infuse nutritive lipidsinto thestomach.Deafferentated micefailed to gradually increaseintra-gastric feeding over daily sessions:N = 6 in each group,Two-way mixed model ANOVA,main effect ofsessionF[9,90] = 2.6, p = 0.008; maineffect of vagal gut deafferentation F[1,10]= 30.6, p 0.001; session3deafferentationinteractioneffect F[9,90] = 13.8,*p 0.0001.(FF) Caspase-induced vagal deafferentation of the gut fails to disrupt aversion learning to ﬂavors paired to intra-gastric malaise agents (LiCl) versus ﬂavors pairedto intra-gastric vehicle; N = 6 in each group, Two-way mixed model ANOVA, main effect of intra-gastric infusate F[1,10] = 461.3, p 0.0001; main effect of vagalgut deafferentation F[1,10] 0.1, p 0.99; Infusate 3deafferentation interaction effect F[1,10] = 0.05, p = 0.82.(GG) Intake for each ﬂavor during the one-session ﬂavor avoidance learning after caspase-induced vagal deafferentation of the gut. Two-way mixed modelANOVA; Main effect of deafferentation F[1,10] = 4.0, p 0.07, Main effect of treatment F[1,10] = 95.0, p 0.001; deafferentation 3treatment interaction effectF[1,10] = 0.48, p = 0.5.(HH) Caspase-induced vagal deafferentation of the gut abolishes dopamine release in dorsal striatum induced by intra-gastric infusions of nutritive lipids; N = 5 ineach group, Two-way mixed model ANOVA, sampling time 3deafferentation interaction effect F[15,120] = 5.2, *p = 0 0.001.(II) Caspase-induced vagal deafferentation of the gut abolished the suppressive effects of CCK on food intake: N = 5 in each group, main effect of CCK F[1,8] =27.7, p = 0.001; main effect of deafferentation F[1,8] = 1.1, p = 0.32; CCK 3deafferentation interaction effect F[1,8] = 13.0, *p = 0.007. S. Caspase- induced vagaldeafferentation of the gut abolishes dopamine release in dorsal striatum induced by intra -gastric infusions of nutritive lipids; N = 5 in each group, Two-way mixedmodel ANOVA, sampling time 3deafferentation interaction effect F[15,120] = 5.2, *p = 0 0.001.(JJ and KK) Caspase-induced vagal deafferentation of the gut abolishes CCK-induced Fos expression in both NTS (JJ, control left panel; deafferentated rightpanel) and PBNdl (KK, control left panel; deafferentated right panel).(LL) Quantiﬁcation of Fos+ positive cells. N = 5, one-way ANOVAs PBN: F[1,8] = 28.2, p = 0.001; NTS: F[1,8] = 27.18, p = 0.001. AP =area postrema; DMV = dorsalmotor nucleus of the vagus; NTS = nucleus of the solitary tract; SNc =Substantia nigra, pars compacta; VTA = ventral tegmental area; PAG = periaqueductal graymatter; PBNdl = parabrachial nucleus, dorsolateral part; PBNel = parabrachial nucleus, externolateral part; PBNm = parabrachial nucleus, medial part; PPTg =pedunculopontine tegmental nucleus. Bar = 100mM. Data reported as mean ±SEM.Citations (25)References (6)... Specifically, calcium imaging of vagal ganglia showed that GLP1R-expressing neurons are selectively activated by stomach stretch, whereas perfusion of nutrients or high osmolar solutions into the small intestine activates GPR65-expressing neurons (Tan et al., 2020;Williams et al., 2016). Additionally, acute organ-or cell-type-specific stimulation of vagal afferents has been shown to be sufficient to alter food intake (Bai et al., 2019;Chen et al., 2020;Han et al., 2018). Opto-or chemogenetic stimulation of uppergut-innervating, GLP1R-expressing, or oxytocin-receptor-expressing vagal afferents reduced feeding (Bai et al., 2019;Brierley et al., 2021;Han et al., 2018), whereas chemogenetically stimulating vagal afferents that synaptically engage tyrosine-hydroxylase-expressing neurons in the nucleus of the solitary tract (NTS) increased feeding . ...... Additionally, acute organ-or cell-type-specific stimulation of vagal afferents has been shown to be sufficient to alter food intake (Bai et al., 2019;Chen et al., 2020;Han et al., 2018). Opto-or chemogenetic stimulation of uppergut-innervating, GLP1R-expressing, or oxytocin-receptor-expressing vagal afferents reduced feeding (Bai et al., 2019;Brierley et al., 2021;Han et al., 2018), whereas chemogenetically stimulating vagal afferents that synaptically engage tyrosine-hydroxylase-expressing neurons in the nucleus of the solitary tract (NTS) increased feeding . ...... We next tested whether activation of GLP1R and GPR65 vagal afferents increases neural activity in the lateral parabrachial nucleus (PB). This possibility is of interest because NTS and AP neurons project to and synaptically engage PB neurons that control feeding behavior (Campos et al., 2016;Carter et al., 2013;Han et al., 2018;Kim et al., 2020;Roman et al., 2016;Zhang et al., 2021). We found that GLP1R vagal afferent stimulation increased neuronal activity in the external lateral part of the PB (PBe; Figures 3F and 3G). ...Gut-brain communication by distinct sensory neurons differently controls feeding and glucose metabolismArticleFull-text availableMay 2021CELL METAB Diba Borgmann Elisa Ciglieri Nasim Biglari Henning FenselauSensory neurons relay gut-derived signals to the brain, yet the molecular and functional organization of distinct populations remains unclear. Here, we employed intersectional genetic manipulations to probe the feeding and glucoregulatory function of distinct sensory neurons. We reconstruct the gut innervation patterns of numerous molecularly defined vagal and spinal afferents and identify their downstream brain targets. Bidirectional chemogenetic manipulations, coupled with behavioral and circuit mapping analysis, demonstrated that gut-innervating, glucagon-like peptide 1 receptor (GLP1R)-expressing vagal afferents relay anorexigenic signals to parabrachial nucleus neurons that control meal termination. Moreover, GLP1R vagal afferent activation improves glucose tolerance, and their inhibition elevates blood glucose levels independent of food intake. In contrast, gut-innervating, GPR65-expressing vagal afferent stimulation increases hepatic glucose production and activates parabrachial neurons that control normoglycemia, but they are dispensable for feeding regulation. Thus, distinct gut-innervating sensory neurons differentially control feeding and glucoregulatory neurocircuits and may provide specific targets for metabolic control.ViewShow abstract... We describe a practical protocol for locating and accessing the mouse jugular-nodose ganglia in vivo, including instructions for intraganglionic injections and postperfusion dissection. For complete details on the use and execution of this protocol, please refer to Han et al. (2018). ...... Interestingly, several studies indicate that gut-innervating, nutrient-sensing nodose neurons express Cckar/Cckbr and Glp1r (Diepenbroek et al., 2017, Kupari et al., 2019. Jugular-nodose peripheral terminals innervate visceral organs, and their central terminals reach the medulla oblongata (i.e., nucleus tractus solitarius, area postrema, and paratrigeminal nucleus) (Han et al., 2018, McGovern et al., 2015, Altschuler et al., 1989, Kim et al., 2020. ...... We first describe how to safely perform intra-ganglionic injections (Part I). We use as an example the delivery of CCK-conjugated saporin (CCK-SAP), a neurotoxin that can be used to specifically ablate gut-innervating JNG neurons (Han et al., 2018). However, the same protocol can be equally Figure 1. ...Dissection and surgical approaches to the mouse jugular-nodose gangliaArticleFull-text availableJun 2021 Wenfei Han Ivan E de AraujoThe jugular-nodose ganglia contain the sensory peripheral neurons of the vagus nerve, linking visceral organs to the medulla oblongata. Accessing these ganglia in smaller animals without damaging the vascular and neural structures may be challenging, as ganglionic fibers imbed deeply into the carotid sheath, and vagal parasympathetic fibers cross through the interior of the ganglia. We describe a practical protocol for locating and accessing the mouse jugular-nodose ganglia in vivo, including instructions for intraganglionic injections and postperfusion dissection.For complete details on the use and execution of this protocol, please refer to Han et al. (2018).ViewShow abstract... A growing number research has made an emphasis on the bidirectional communication network between gut microbiota and the CNS: the microbiota-gut-brain axis the (Muller et al., 2020). Physically, connections between the gut and brain modulate intestinal functions such as nutrient absorption and motility (Tuganbaev et al., 2020;Williams et al., 2016) and brain-wired feeding behavior (Han et al., 2018) as well as fetal neurodevelopment (Vuong et al., 2020). Nowadays, microorganisms are found to influence CNS process at least via vagus nerve (Muller et al., 2020) and through modulation of the immune system (Fung, Olson, Hsiao, 2017), along with synthesis of a number of neurotransmitters (Jameson Hsiao, 2018) and production of the microbial metabolites, SCFAs (Dalile, Van Oudenhove, Vervliet, Verbeke, 2019). ...Dietary nutrition for neurological disease therapy: Current status and future directionsArticleFull-text availableJan 2021PHARMACOL THERAPEUT Xiao-Yuan Mao Xixi Yin Qi-Wen Guan Wei-Lin JinEditor: J.P. Hardwick Keywords: Dietary nutrient Neurological disease Metabolism Epigenetics Immunity Therapy Adequate food intake and relative abundance of dietary nutrients have undisputed effects on the brain function. There is now substantial evidence that dietary nutrition aids in the prevention and remediation of neurologic symptoms in diverse pathological conditions. The newly described influences of dietary factors on the alterations of mitochondrial dysfunction, epigenetic modification and neuroinflammation are important mechanisms that are responsible for the action of nutrients on the brain health. In this review, we discuss the state of evidence supporting that distinct dietary interventions including dietary supplement and dietary restriction have the ability to tackle neurological disorders using Alzheimer s disease, Parkinson s disease, stroke, epilepsy, traumatic brain injury, amyotrophic lateral sclerosis, Huntington s disease and multiple sclerosis as examples. Additionally, it is also highlighting that diverse potential mechanisms such as metabolic control, epigenetic modification, neu-roinflammation and gut-brain axis are of utmost importance for nutrient supply to the risk of neurologic condition and therapeutic response. Finally, we also highlight the novel concept that dietary nutrient intervention reshapes metabolism-epigenetics-immunity cycle to remediate brain dysfunction. Targeting metabolism-epigenetics-immunity network will delineate a new blueprint for combating neurological weaknesses.ViewShow abstract... Mice fed high-fat diets do not show the caloriedependent DA effluxes seen in mice fed low-fat diets and this high-fat-induced DA deficiency is restored by the dietary satiety messenger oleoylethanolamine (Tellez et al., 2013). More recently, these same investigators have identified the neural circuit for gut-induced reward by showing that optical activation of the right nodose ganglion causes release of DA in the striatum, sustains self-stimulation behavior, and conditions a place preference [conditioned place preference (CPP)] that maps to populations of well known reward neurons in the nigro-striatal pathway (Han et al., 2018). In a related study, Fernandes et al. (2020) demonstrated that the intragastric administration of sucrose sustains self-administration and increases the activity of VTA-DA neurons via the hepatic branch of the vagus nerve. ...Evidence for Modulation of Substance Use Disorders by the Gut Microbiome: Hidden in Plain SightArticleApr 2021PHARMACOL REVMariana Angoa-Pérez Donald KuhnThe gut microbiome modulates neuro-chemical function and behavior and has been impli-cated in numerous central nervous system (CNS) diseases, including developmental, neurodegenerative, and psychiatric disorders. Substance use disorders (SUDs) remain a serious threat to the public well-being, yet gut microbiome involvement in drug abuse has received very little attention. Studies of the mechanisms underlying SUDs have naturally focused on CNS reward circuits. However, a significant body of research has accumulated over the past decade that has unwittingly provided strong support for gut microbiome participation in drug reward. β-Lactam antibiotics have been employed to increase glutamate transporter expression to reverse relapse-induced release of glutamate. Sodium butyrate has been used as a histone deacetylase inhibitor to prevent drug-induced epigenetic alterations. High-fat diets have been used to alter drug reward because of the extensive overlap of the circuitry mediating them. This review article casts these approaches in a different light and makes a compelling case for gut microbiome modulation of SUDs. Few factors alter the structure and composition of the gut microbiome more than antibiotics and a high-fat diet, and butyrate is an endogenous product of bacterial fermentation. Drugs such as cocaine, alcohol, opiates, and psychostimulants also modify the gut microbiome. Therefore, their effects must be viewed on a complex background of cotreatment-induced dysbiosis. Consideration of the gut microbiome in SUDs should have the beneficial effects of expanding the understanding of SUDs and aiding in the design of new therapies based on opposing the effects of abused drugs on the host’s commensal bacterial community. © 2021, American Society for Pharmacology and Experimental Therapy. All rights reserved.ViewShow abstract... Our findings here demonstrate that, in addition to spinal sensory nerves, EEC-vagal signaling is an important pathway for transmitting specific gut microbial signals to the CNS. The vagal ganglia project directly onto the hindbrain, and that vagal-hindbrain pathway has key roles in appetite and metabolic regulation (Grill and Hayes, 2009;Han et al., 2018;Travagli et al., 2006;Berthoud et al., 2006). Our findings raise the possibility that certain tryptophan catabolites, including indole, may directly impact these processes as well as emotional behavior and cognitive function (Jaglin et al., 2018). ...Enteroendocrine cells sense bacterial tryptophan catabolites to activate enteric and vagal neuronal pathwaysArticleDec 2020CELL HOST MICROBELihua YeMunhyung BaeChelsi D. Cassilly John Franklin RawlsThe intestinal epithelium senses nutritional and microbial stimuli using epithelial sensory enteroendocrine cells (EEC). EECs communicate nutritional information to the nervous system, but whether they also relay signals from intestinal microbes remains unknown. Using in vivo real-time measurements of EEC and nervous system activity in zebrafish, we discovered that the bacteria Edwardsiella tarda activate EECs through the receptor transient receptor potential ankyrin A1 (Trpa1) and increase intestinal motility. Microbial, pharmacological, or optogenetic activation of Trpa1⁺EECs directly stimulates vagal sensory ganglia and activates cholinergic enteric neurons by secreting the neurotransmitter 5-hydroxytryptamine (5-HT). A subset of indole derivatives of tryptophan catabolism produced by E. tarda and other gut microbes activates zebrafish EEC Trpa1 signaling. These catabolites also directly stimulate human and mouse Trpa1 and intestinal 5-HT secretion. These results establish a molecular pathway by which EECs regulate enteric and vagal neuronal pathways in response to microbial signals.ViewShow abstract... The authors find that bilateral subdiaphragmatic vagotomy abrogates the ability of L. reuteri to restore sociability and reciprocal social interaction in Shank3B À/À mice ( Figure 1). This is consistent with recent findings that signaling through intestinal afferent sensory neurons mediates the effects of gut endocrine, immune, and dietary sensory signals on brain activity (Han et al., 2018;Kaelberer et al., 2018). Together, these results reveal that the vagus nerve is required for social behavioral modification by L. reuteri, and further suggest that future studies examining how select microbes and microbial factors modulate intestinal sensory neuronal activity may uncover novel molecular targets for altering social behavior. ...Gut Microbes Join the Social NetworkArticleJan 2019NEURON Helen Vuong Elaine HsiaoThe gut microbiome is increasingly implicated in the regulation of social behavior across model organisms. In this issue of Neuron, Sgritta et al. (2018) examine the role of the gut microbiome in social reward circuits and sociability in three mouse models of autism spectrum disorder.ViewShow abstractMICROBIAL METABOLITES AND THE VAGAL AFFERENT PATHWAY IN THE CONTROL OF FOOD INTAKEArticleAug 2021PHYSIOL BEHAVDanielle ZumpanoHelen E RaybouldThe gut microbiota is able to influence overall energy balance via effects on both energy intake and expenditure, and is a peripheral target for potential obesity therapies. However, the precise mechanism by which the gut microbiota influences energy intake and body weight regulation is not clear. Microbes use small molecules to communicate with each other; some of these molecules are ligands at mammalian receptors and this may be a mechanism by which microbes communicate with the host. Here we briefly review the literature showing beneficial effects of microbial metabolites on food intake regulation and examine the potential role for vagal afferent neurons, the gut-brain axis.ViewShow abstractMidbrain and Lateral Nucleus Accumbens Dopamine Depletion Affects Free-choice High-fat high-sugar Diet Preference in Male RatsArticleMay 2021NEUROSCIENCE Anil Joshi Fanny FaivreSusanne Eva la FleurMichel BarrotDopamine influences food intake behavior. Reciprocally, food intake, especially of palatable dietary items, can modulate dopamine-related brain circuitries. Among these reciprocal impacts, it has been observed that an increased intake of dietary fat results in blunted dopamine signaling and, to compensate this lowered dopamine function, caloric intake may subsequently increase. To determine how dopamine regulates food preference we performed 6-hydroxydopamine (6-OHDA) lesions, depleting dopamine in specific brain regions in male Sprague Dawley rats. Food preference was assessed by providing the rats with free choice access to control diet, fat, 20% sucrose and tap water. Rats with midbrain lesions targeting the substantia nigra (which is also a model of Parkinson’s disease) consumed fewer calories, as reflected by a decrease in control diet intake, but they surprisingly displayed an increase in fat intake, without change in the sucrose solution intake compared to sham animals. To determine which of the midbrain dopamine projections may contribute to this effect, we next compared the impact of 6-OHDA lesions of terminal fields, targeting the dorsal striatum, the lateral nucleus accumbens and the medial nucleus accumbens. We found that 6-OHDA lesion of the lateral nucleus accumbens, but not of the dorsal striatum or the medial nucleus accumbens, led to increased fat intake. These findings indicate a role for lateral nucleus accumbens dopamine in regulating food preference, in particular the intake of fat.ViewShow abstractAuricular Vagal Nerve Stimulation Improves Constipation by Enhancing Colon Motility via the Central-Vagal Efferent Pathway in Opioid-Induced Constipated RatsArticleApr 2021NeuromodulationYiling ZhangTao LuYan Meng Jiande ChenObjectives: Constipation and opioid-induced constipation (OIC) are common with limited treatment options. We investigated whether a noninvasive method of auricular vagal nerve stimulation (aVNS) could be used for treating OIC and explored its potential mechanisms and neural pathways in a rodent model of OIC.Materials and methods: Sprague-Dawley were chronically implanted with one pair of auricular electrodes for aVNS. Sixteen rats were treated with loperamide for a week while another 16 rats received bilateral vagotomy, then randomly treated with aVNS or sham-aVNS for a week. In addition, eight normal rats were implanted with a polyethylene catheter in the proximal colon for assessing whole colon transit.Results: 1) The number of fecal pellets and water content in feces increased after aVNS, compared with sham-aVNS. 2) aVNS accelerated colon transit and whole gut transit, compared with sham-aVNS. 3) In colon tissues, aVNS increased the protein expression of choline acetyltransferase, glial cell line-derived neurotrophic factor and the c-kit expression in myenteric interstitial cells of Cajal but decreased the protein expression of neural nitric oxide synthase (p 0.05 for all, vs. sham-VNS). 4) The prokinetic effects of aVNS were abolished by both subdiaphragmatic vagotomy and atropine. 5) aVNS increased the c-fos expression in both nucleus tractus solitarius and dorsal motor nucleus of vagus, and increased vagal efferent activity (p 0.05, vs. sham-VNS).Conclusions: aVNS improves OIC by enhancing colon motility and restoring enteric neural functions mediated via the central and vagal efferent pathway.ViewShow abstractInnate gut microbiota predisposes to high alcohol consumptionArticleFull-text availableJan 2021ADDICT BIOL Fernando E Ezquer María Elena QuintanillaFrancisco Moya-Flores Yedy IsraelGut microbiota is known to be transferred from the mother to their offspring. This study determines whether the innate microbiota of rats selectively bred for generations as high alcohol drinkers play a role in their alcohol intake. Wistar‐derived high‐drinker UChB rats (intake 10‐g ethanol/kg/day) administered nonabsorbable oral antibiotics before allowing access to alcohol, reducing their voluntary ethanol intake by 70%, an inhibition that remained after the antibiotic administration was discontinued. Oral administration of Lactobacillus rhamnosus Gorbach–Goldin (GG) induced the synthesis of FGF21, a vagal β‐Klotho receptor agonist, and partially re‐invoked a mechanism that reduces alcohol intake. The vagus nerve constitutes the main axis transferring gut microbiota information to the brain (\"microbiota‐gut‐brain” axis). Bilateral vagotomy inhibited rat alcohol intake by 75%. Neither antibiotic treatment nor vagotomy affected total fluid intake. A microbiota‐mediated marked inflammatory environment was observed in the gut of ethanol‐naïve high‐drinker rats, as gene expression of proinflammatory cytokines (TNF‐α; IL‐6; IL‐1β) was significantly reduced by nonabsorbable antibiotic administration. Gut cytokines are known to activate the vagus nerve, while vagal activation induces pro‐rewarding effects in nucleus accumbens. Both alcoholics and alcohol‐preferring rats share a marked preference for sweet tastes—likely an evolutionary trait to seek sweet fermented fruits. Saccharin intake by UChB rats was inhibited by 75%–85% by vagotomy or oral antibiotic administration, despite saccharin‐induced polydipsia. Overall, data indicate that the mechanisms that normally curtail heavy drinking are inhibited in alcohol‐preferring animals and inform a gut microbiota origin. Whether it applies to other mammals and humans merits further investigation.ViewShow abstractShow moreRight panel: Quantification of supra-threshold events during stomach electromyogram recordings performed under optogenetic stimulation. n = 9-HH. Control experiments. Optical fibers were implanted above the NTS (in the area more intensely innervated by R-NG terminals) in miceLeftLeft, upper panel: Representative electromyogram trace of stomach musculature upon light stimulation of L-NG DIO-ChR2-EYFP-positive neurons. The trace shows that light pulses elicited no gut motility responses (laser pulses marked as blue bars on top). Right panel: Quantification of supra-threshold events during stomach electromyogram recordings performed under optogenetic stimulation. n = 9-HH. Control experiments. Optical fibers were implanted above the NTS (in the area more intensely innervated by R-NG terminals) in mice injected with ChR2 into the L-NG.Left panel: Representative heatmap showing the pre-test baseline (upper) and on-line place preference (lower)PbnelPBNel[VGlut2]/CeL/C optical stimulation induces place avoidance. Left panel: Representative heatmap showing the pre-test baseline (upper) and on-line place preference (lower). Right panel: Place avoidance for laser-paired side, N = 6; paired t test t[5] = 12.3, *p = 0.001.Dark green points represents laser OFF, and light green line represents laser ON, with blue area indicating days in which laser was ON. N = 5, two-way RM-ANOVA laser 3 session interaction effectPbnelPBNel[VGat] optical stimulation during ingestion of 5% IntraLipid. After daily baseline sessions 1-3, intake is reduced during laser ON sessions 4-6, and return to baseline levels was delayed on post-laser sessions 7-9. Dark green points represents laser OFF, and light green line represents laser ON, with blue area indicating days in which laser was ON. N = 5, two-way RM-ANOVA laser 3 session interaction effect F[8,32] = 9.5, *p 0.001.N = 6; Two-way RM-ANOVA, Main effect of poking on the laser-paired hole versus inactive hole F[1,5] = 2.5,p = 0.17. Mice varied number of responses over daily sessions: Main effect of sessionPbnelPBNel[VGlut2]/CeL/C optical stimulation fails to sustain self-stimulation behavior. N = 6; Two-way RM-ANOVA, Main effect of poking on the laser-paired hole versus inactive hole F[1,5] = 2.5,p = 0.17. Mice varied number of responses over daily sessions: Main effect of session, F[2,10] = 10.4, p = 0.004. However, these changes in response rates were not specific to poking on the laser-paired hole: Laser 3 session interaction effect: F[2,10] = 0.8, p = 0.44. No effects wereCeL/C optical stimulation induces moderate flavor avoidance. Post-conditioning flavor preferences for laser-paired flavors N = 6PbnelPBNel[VGlut2]/CeL/C optical stimulation induces moderate flavor avoidance. Post-conditioning flavor preferences for laser-paired flavors N = 6; paired t testAfter daily baseline sessions 1-3, intake is reduced during laser ON sessions 4-6, and immediately returned to baseline on post-laser sessions 7-9PbnelPBNel[VGlut2]/CeL/C optical stimulation during ingestion of 5% IntraLipid. After daily baseline sessions 1-3, intake is reduced during laser ON sessions 4-6, and immediately returned to baseline on post-laser sessions 7-9. N = 6, two-way RM-ANOVAAdvertisementRecommendationsDiscover moreProjectNeurobiology of Feeding Wenfei Han Luis A. Tellez Xiaobing Zhang[...]Li YanView projectProjectModulation of radula opening in feeding network states. Matthew H Perkins Klaudiusz R Weiss Elizabeth C CropperView projectProjectBoth emotional and cognitive alterations by orofacial chronic neuropathic pain Isaac Obed Perez Nadia Estefanía Gutiérrez Castañeda Claudia Daniela Montes-AngelesExperimental evaluation of chronic pain s effects in emotional and cognitive processing. View projectArticleL’anhédonie dans la dépressionSeptember 2013 · L Encéphale Raphaël Gaillard David Gourion P.-M. LlorcaAnhedonia, or markedly diminished interest or pleasure, is a hallmark symptom of major depression, schizophrenia, and other neuropsychiatric disorders. The term \"anhedonia” was introduced by the French psychologist Ribot in 1896 to describe the counterpart to analgesia in his patients, for which \"it was impossible to find the least pleasure”. Over the last decades, the clinical definition of ... [Show full abstract] anhedonia has remained relatively unchanged, but recently, behavioral neurosciences have significantly changed our knowledge of reward-related processes. Now, the construct of anhedonia reflects deficits in hedonic capacity, and is closely linked to the processes of reward valuation, decision-making, anticipation, and motivation. The neural circuits underlying these reward-related mechanisms include essentially the ventral striatum and prefrontal cortical regions. Here, we review the clinical concepts, neural bases and psychopharmacological data related to the deficits of hedonia in depression. Understanding anhedonia will facilitate diagnosis and treatment management.Read moreArticleFull-text availableAdult zebrafish as a model organism for behavioral geneticsAugust 2010 · BMC Neuroscience Will Norton Laure Bally-CuifRecent research has demonstrated the suitability of adult zebrafish to model some aspects of complex behaviour. Studies of reward behaviour, learning and memory, aggression, anxiety and sleep strongly suggest that conserved regulatory processes underlie behaviour in zebrafish and mammals. The isolation and molecular analysis of zebrafish behavioural mutants is now starting, allowing the ... [Show full abstract] identification of novel behavioural control genes. As a result of this, studies of adult zebrafish are now helping to uncover the genetic pathways and neural circuits that control vertebrate behaviour.View full-textArticleFull-text availableReward signaling in a recurrent circuit of dopaminergic neurons and peptidergic Kenyon cellsJuly 2019 · Nature Communications Radostina Lyutova Mareike SelchoMaximilian Pfeuffer[...] Dennis PaulsDopaminergic neurons in the brain of the Drosophila larva play a key role in mediating reward information to the mushroom bodies during appetitive olfactory learning and memory. Using optogenetic activation of Kenyon cells we provide evidence that recurrent signaling exists between Kenyon cells and dopaminergic neurons of the primary protocerebral anterior (pPAM) cluster. Optogenetic activation ... [Show full abstract] of Kenyon cells paired with odor stimulation is sufficient to induce appetitive memory. Simultaneous impairment of the dopaminergic pPAM neurons abolishes appetitive memory expression. Thus, we argue that dopaminergic pPAM neurons mediate reward information to the Kenyon cells, and in turn receive feedback from Kenyon cells. We further show that this feedback signaling is dependent on short neuropeptide F, but not on acetylcholine known to be important for odor-shock memories in adult flies. Our data suggest that recurrent signaling routes within the larval mushroom body circuitry may represent a mechanism subserving memory stabilization.View full-textArticleDefining biotypes for depression and anxiety based on large-scale circuit dysfunction: A theoretical...January 2017 · Depression and Anxiety Leanne M WilliamsComplex emotional, cognitive and self-reflective functions rely on the activation and connectiv-ity of large-scale neural circuits. These circuits offer a relevant scale of focus for conceptualizing a taxonomy for depression and anxiety based on specific profiles (or biotypes) of neural circuit dysfunction. Here, the theoretical review first outlines the current consensus as to what constitutes ... [Show full abstract] the organization of large-scale circuits in the human brain identified using parcellation and meta-analysis. The focus is on neural circuits implicated in resting reflection (default mode), detection of salience, affective processing ( threat and reward ), attention, and cognitive control. Next, the current evidence regarding which type of dysfunctions in these circuits characterize depression and anxiety disorders is reviewed, with an emphasis on published meta-analyses and reviews of circuit dysfunctions that have been identified in at least two well-powered case:control studies. Grounded in the review of these topics, a conceptual framework is proposed for considering neural circuit-defined biotypes. In this framework, biotypes are defined by profiles of extent of dysfunction on each large-scale circuit. The clinical implications of a biotype approach for guiding classification and treatment of depression and anxiety is considered. Future research directions will develop the validity and clinical utility of a neural circuit biotype model that spans diagnostic categories and helps to translate neuroscience into clinical practice in the real world.Read moreArticleFull-text availableDentate network activity is necessary for spatial working memory by supporting CA3 sharp-wave ripple...February 2018 · Nature NeuroscienceTakuya Sasaki Verónica C PiattiErnie Hwaun[...]Jill K LeutgebComplex spatial working memory tasks have been shown to require both hippocampal sharp-wave ripple (SWR) activity and dentate gyrus (DG) neuronal activity. We therefore asked whether DG inputs to CA3 contribute to spatial working memory by promoting SWR generation. Recordings from DG and CA3 while rats performed a dentate-dependent working memory task on an eight-arm radial maze revealed that the ... [Show full abstract] activity of dentate neurons and the incidence rate of SWRs both increased during reward consumption. We then found reduced reward-related CA3 SWR generation without direct input from dentate granule neurons. Furthermore, CA3 cells with place fields in not-yet-visited arms preferentially fired during SWRs at reward locations, and these prospective CA3 firing patterns were more pronounced for correct trials and were dentate-dependent. These results indicate that coordination of CA3 neuronal activity patterns by DG is necessary for the generation of neuronal firing patterns that support goal-directed behavior and memory.View full-textDiscover the world s researchJoin ResearchGate to find the people and research you need to help your work.Join for free ResearchGate iOS AppGet it from the App Store now.InstallKeep up with your stats and moreAccess scientific knowledge from anywhere orDiscover by subject areaRecruit researchersJoin for freeLoginEmail Tip: Most researchers use their institutional email address as their ResearchGate loginPasswordForgot password? 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