ARTICLE DOI: 10.1038/s41467-018-04639-1 OPEN Gut vagal sensory signaling regulates hippocampus function through multi-order pathways 1 2,3 1,2 1 1 2 Andrea N. Suarez , Ted M. Hsu , Clarissa M. Liu , Emily E. Noble , Alyssa M. Cortella , Emily M. Nakamoto , 4 5,6 1,2,4 Joel D. Hahn , Guillaume de Lartigue & Scott E. Kanoski The vagus nerve is the primary means of neural communication between the gastrointestinal (GI) tract and the brain. Vagally mediated GI signals activate the hippocampus (HPC), a brain region classically linked with memory function. However, the endogenous relevance of GI- derived vagal HPC communication is unknown. Here we utilize a saporin (SAP)-based lesioning procedure to reveal that selective GI vagal sensory/afferent ablation in rats impairs HPC-dependent episodic and spatial memory, effects associated with reduced HPC neuro- trophic and neurogenesis markers. To determine the neural pathways connecting the gut to the HPC, we utilize monosynaptic and multisynaptic virus-based tracing methods to identify the medial septum as a relay connecting the medial nucleus tractus solitarius (where GI vagal afferents synapse) to dorsal HPC glutamatergic neurons. We conclude that endogenous GI- derived vagal sensory signaling promotes HPC-dependent memory function via a multi-order brainstem–septal pathway, thereby identifying a previously unknown role for the gut–brain axis in memory control. 1 2 Human and Evolutionary Biology Section, Department of Biological Sciences, University of Southern California, Los Angeles, California, USA. Neuroscience Graduate Program, University of Southern California, Los Angeles, California, USA. Department of Psychology, University of Illinois at Chicago, Chicago, 4 5 Illinois, USA. Neurobiology Section, Department of Biological Sciences, University of Southern California, Los Angeles, California, USA. The John B. Pierce Laboratory, New Haven, Connecticut, USA. Department of Cellular and Molecular Physiology, Yale Medical School, New Haven, Connecticut, USA. Correspondence and requests for materials should be addressed to G.L. (email: firstname.lastname@example.org) or to S..K. (email: email@example.com) NATURE COMMUNICATIONS (2018) 9:2181 DOI: 10.1038/s41467-018-04639-1 www.nature.com/naturecommunications 1 | | | 1234567890():,; ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04639-1 nergy balance and metabolic-relevant communication memory and contextual episodic memory) ; (2) discrimination between the gastrointestinal (GI) tract and the brain is learning based on interoceptive energy status cues (food restric- Emediated largely by the vagus nerve. Vagal afferent/sensory tion vs. satiety) ; and (3) social transmission of olfactory-related information is received ﬁrst in the brain within the medial food cues . To dissociate between the role of GI vagal sensory vs. nucleus of the solitary tract (mNTS) in the caudal brainstem and motor signaling on HPC-dependent memory, we utilized total then relayed to various hindbrain and forebrain regions via subdiaphragmatic vagotomy (SDV; eliminates all GI vagal affer- ascending neural pathways . Neurons in the hippocampus ents and efferents) and a novel rodent surgical approach for (HPC), a brain region traditionally linked with learning and selective GI vagal deafferentation in which a SAP conjugated to memory control and more recently with feeding behavior , are cholecystokinin (CCK-SAP) is injected into the nodose ganglia activated by direct vagal nerve stimulation and by GI vagally (overview of approaches in Fig. 1a, b). This recently established mediated signals such as mechanical distension of the stomach procedure eliminates ~ 80% of GI-derived vagal sensory input to 3–5 and intestinal nutrient infusion . In addition, rats with selective the brain while leaving intact all brain-to-gut vagal motor sig- HPC lesions are impaired in utilizing interoceptive hunger and naling, and supradiaphragmatic and colonic vagal sensory sig- 6 17 satiety cues to guide learned anticipatory appetitive outcomes , naling . Results show that vagal gut–brain sensory signaling is suggesting that the HPC functionally integrates GI energy required for hippocampal-dependent learning processes based on balance-relevant cues. Unknown is whether feeding-relevant GI external and visuospatial cues, effects accompanied by reduced vagal afferent signaling endogenously impacts cognitive and hippocampal expression of neurotrophic (BDNF) and neurogenic mnemonic processes that are regulated by the HPC. (doublecortin, DCX) markers. Using monosynaptic and multi- Consistent with a role for vagal signaling in memory function, synaptic virus-based neural pathway tracing methods, we also 7, 8 vagus nerve stimulation enhances memory , facilitates HPC identiﬁed a multi-order pathway connecting the medullary mNTS neurogenesis, and increases HPC expression of brain-derived to the dorsal HPC via medial septum (MS) input to HPC gluta- 9, 10 neurotrophic factor (BDNF) , a neurotrophin that promotes matergic neurons. neuronal survival and differentiation, as well as synaptic plasti- city . These ﬁndings suggest that the vagus nerve promotes neurogenic and neurotrophic signaling. However, these ﬁndings Results involve non-physiological electrical stimulation of the cervical SDV and CCK-SAP impair contextual episodic memory. Novel vagus nerve. The endogenous relevance of vagal signaling, espe- object in context (NOIC) learning is a rodent model of contextual cially gut-innervating vagal afferent pathways, to mnemonic and episodic memory. During day 1 of training, SDV and sham cognitive control is poorly understood. Furthermore, the neural groups exhibited similar object discrimination indices (DIs; a pathways through which vagally mediated energy-state signals are measure of exploration time of both objects, Fig. 2a, left), indi- transmitted between the GI tract and hippocampal neurons cating that baseline preference for objects A and B did not differ remains to be fully understood. The mNTS, where GI-derived by group. On test day, SDV animals had impaired contextual vagal sensory inputs synapse, sends projections to many brain- episodic memory, demonstrated by a signiﬁcantly reduced DI 12–14 stem and forebrain sites, but none directly to the HPC . Thus, relative to sham animals (Fig. 2a, right; a DI above 0.50 means the neural communication between the gut and the HPC must animals spent more time exploring the novel object for the test involve a yet unidentiﬁed multi-order neural pathway. context). Repeated-measures analysis of variance (ANOVA) The present study investigated the endogenous role of GI- analyses across days revealed a signiﬁcant day × group interaction derived (subdiaphragmatic) vagus nerve signaling on a variety of (F[1,13] = 5.564, p = 0.0347), with Newman–Keuls’ post hoc HPC-dependent memory processes that involve the following: (1) analyses conﬁrming a signiﬁcant sham vs. SDV group difference processing of external visuospatial stimuli (i.e., spatial working on day 3 (p = 0.0047) but not on day 1. ab Total subdiaphragmatic vagotomy (SDV) CCK-saporin (CCK-SAP) nodose ganglia injections mNTS mNTS DMX DMX Vagal sensory input to mNTS Vagal motor output from DMX ~20% vagal sensory input intact Fig. 1 Schematic illustration of subdiaphragmatic vagus nerve ablative disconnection methods. a Classic total subdiaphragmatic vagotomy (SDV) surgical method consists of lesioning the dorsal and ventral subdiaphragmatic vagus nerve, eliminating 100% of vagal afferent (sensory) and efferent (motor) signaling below the diaphragm. b The novel CCK saporin (CCK-SAP) approach consists of nodose ganglia injections of saporin conjugated to cholecystokinin to speciﬁcally ablate ~ 80% of vagal gastrointestinal (GI)-innervating afferent signaling, while leaving 100% of vagal efferent and supradiaphragmatic vagal afferent signaling intact (see ref. ). (DMX dorsal motor nucleus of the vagus nerve, mNTS medial nucleus tractus solitarius). [Cartoon schematic made by authors based on ref. ] 2 NATURE COMMUNICATIONS (2018) 9:2181 DOI: 10.1038/s41467-018-04639-1 www.nature.com/naturecommunications | | | NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04639-1 ARTICLE Analogous to the SDV and sham group, CCK-SAP and SAP the spatial location of an escape hole is constant across two (controls) groups demonstrated similar DIs on day 1 of NOIC consecutive trials per day, but changes each subsequent training training (Fig. 2g, left). On test day, however, the CCK-SAP group day. The index of learning on this task is the difference in the had a signiﬁcantly reduced DI relative to the sham group (Fig. 2g, number of errors (exploration of escape holes that do not contain right). Repeated-measures ANOVA analyses revealed a signiﬁ- the escape box) from trial 2 to trial 1 for each individual training cant day × group interaction (F [1,15] = 6.496, p = 0.0223), with day. Results show that the SDV group was impaired in spatial Newman–Keuls’ post hoc analyses conﬁrming a signiﬁcant SAP working memory performance relative to shams (Fig. 2b, right). vs. CCK-SAP group interaction on day 3 (p = 0.0241) but not on Repeated-measures ANOVA analyses of average difference in day 1. Thus, hippocampal-dependent contextual episodic mem- number of errors from trial 2 to trial 1 revealed signiﬁcant group ory in rats requires intact GI vagal afferent signaling. main effect across the 5 training days (F[1,19] = 6.8565, p = 0.0169). Individual training day analyses showed trends toward a group effect statistical signiﬁcance on Day 2 (F[1,14] = 3.626, p = 0.0776, ANOVA), Day 4 (F[1,14] = 3.842, p = 0.0702, SDV and CCK-SAP impair spatial working memory.We ANOVA), and Day 5 (F[1,14] = 3.555, p = 0.0803, ANOVA) developed a modiﬁed Barnes maze procedure to assess compared with shams (Fig. 2b, left). hippocampal-dependent spatial working memory in rats in which ab Novel object in context Spatial working memory 0.8 4 Sham Sham SDV Sham SDV SDV 0.7 0.6 0 0.5 –2 0.4 –4 –2 0.3 –6 –4 0.2 –8 –6 0.1 –10 –12 –8 Day 1 Day 3 D1 D2 D3 D4 D5 c d Deprivation discrimination pre-surgery training Deprivation discrimination post-surgery testing Group 0+ Group 24+ Group 0+ Group 24+ * 100 90 Sham SDV Sham SDV 60 * * 70 40 50 * 0 hr dep 24 hr dep 1 23456 1 2 3 4 5 6 8-trial blocks 8-trial blocks e f Social transmission of food preference Zero maze 100 5 SDV paired Sham paired 140 25 Sham SDV Sham SDV Sham SDV Sham non-paired SDV non-paired 80 4 * * 70 100 60 3 40 2 20 1 5 0 0 0 0 gh Novel object in context Spatial working memory Saporin Saporin CCK-saporin CCK-saporin 0.8 Saporin 10 4 CCK-saporin 8 0.7 0.6 * 0.5 0.4 –2 –2 0.3 –4 –4 0.2 –6 –6 0.1 –8 0 –10 –8 Day 1 Day 3 D1 D2 D3 D4 D5 NATURE COMMUNICATIONS (2018) 9:2181 DOI: 10.1038/s41467-018-04639-1 www.nature.com/naturecommunications 3 | | | 24 hr dep 0 hr dep 24 hr dep 0 hr dep 24 hr dep 0 hr dep 24 hr dep 0 hr dep 30-min percent paired Anticipatory appetitive responding Discrimination index flavor preference (% of 20-sec epochs w/ a response during the last minute) Discrimination index 30-min cumulative food intake (grams) Anticipatory appetitive responding Difference in # of errors Difference in # of errors (% of 20-sec epochs w/ a response from trial 2 to trial 1 from trial 2 to trial 1 during the last minute) Time in open section (seconds) Average difference in # of errors Average difference in # of errors from trial 2 to trial 1 across training Number of open section entries from trial 2 to trial 1 across training ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04639-1 Similar to the SDV and sham groups, the CCK-SAP group was supported by Newman–Keuls’ post hoc analyses revealing a sig- also impaired in this task relative to SAP controls, with repeated- niﬁcant 0 h vs. 24 h deprivation state interaction for Group 0 + measures ANOVA analyses of average error difference (T2–T1) SDV (p = 0.0028), Group 0 + sham (p = 0.004), Group 24 + SDV revealing a signiﬁcant group main effect across the 5 training days (p = 0.000589), and Group 24 + sham (p = 0.000894). These (F[1,13] = 8.66, p = 0.0114) (Fig. 2h, left). Individual training day results suggest that in the absence of GI vagal signaling via SDV, analyses also indicated a signiﬁcant group effect in error non-vagal cues are sufﬁcient to sustain the learned ability to use difference (T2–T1) on Day 2 (F[1,13] = 6.824, p = 0.0215, interoceptive energy status cues as discriminative stimuli for food ANOVA) relative to sham (Fig. 2h, right). Overall, these ﬁndings reinforcement. indicate that spatial working memory in rats is impaired Social transmission of food preference (STFP) is a following GI vagal afferent ablation. hippocampal-dependent procedure involving social-based learn- ing using olfactory cues . Percent paired ﬂavor preference at testing was above 50% chance for both sham and SDV groups SDV does not affect interoceptive or social learning. Depriva- (Fig. 2e, left). Both sham and SDV group signiﬁcantly tion intensity discrimination learning is a hippocampal- preferred the paired ﬂavored chow to the non-paired ﬂavored dependent procedure in which rats learned to use interoceptive chow when tested 24 h after the social interaction (Fig. 2e, right; energy status cues (0 vs. 24 h food restriction) as discriminative 30 min cumulative food intake). One-way ANOVA analyses stimuli for a forthcoming food reinforcer . Repeated-measures revealed no signiﬁcant SDV vs. sham group effect for 30 min ANOVA over six 8-trial blocks of training (before SDV and sham percent paired ﬂavor preference (F[1,12] = 0.0225, p = 0.883). surgeries) showed that both Group 0 + and Group 24 + learned Paired Student’s t-test analysis indicated a signiﬁcant preference to respond more (anticipatory food cue entries before food pellet for paired vs. non-paired ﬂavored chow for both SDV (p = 0.014) delivery) during the last minute of test sessions under their and sham (p = 0.014) groups. Thus, GI vagal signaling has reinforced compared to non-reinforced food restriction level minimal impact on hippocampal-dependent social olfactory- (Fig. 2c), supported by a signiﬁcant deprivation state × depriva- based learning. tion group interaction (F[1,22] = 135.54, p < 0.0001) and a sig- niﬁcant block × deprivation state × deprivation group interaction (F[5,110] = 13.6535, p < 0.0001). When analyzing each block Neither SDV nor CCK-SAP affect innate anxiety or body individually, Newman–Keuls’ post hoc analyses indicate that weight. The Zero maze procedure is an established rodent model Group 0 + responded signiﬁcantly more under 0 h compared of anxiety-like behavior that is similar to the elevated plus maze with 24 h food deprivation during blocks 3–6 (all ps < 0.0017, procedure. ANOVA revealed no signiﬁcant surgical group main Fig. 2c, left), whereas Group 24 + responded signiﬁcantly more effect between SDV and shams for time spent in the open under 24 h compared with 0 h food deprivation during blocks 2–6 section (F[1,19] = 0.0454, p = 0.833) (Fig. 2f; left) and number of (all ps < 0.000178, Fig. 2c, right). Testing of deprivation intensity open section entries (F[1,19] = 4.861, p = 0.731) (Fig. 2f; right). discrimination retention occurred following recovery from SDV Similarly, ANOVA revealed no signiﬁcant surgical group main and sham surgeries. Results conﬁrmed that SDV lesions had no effect between CCK-SAP vs. SAP for: time in open section impact on retention of this type of interoceptive-based dis- (F[1, 15] = 0.0103, p = 0.92) and number of open section entries crimination. In Fig. 2d, the effects of total SDV vs. sham surgery (F[1,15] = 0, p = 1.0; groups had equal means) in the zero maze on food cup entry during the last minute of each test session test of innate anxiety (Supplementary Fig. 1a), as well as center performance when both Groups 0 + and 24 + were tested under zone distance (F[1,15] = 0.198, p = 0.663), number of center zone 0 h and 24 h food deprivation are shown. Repeated-measures entries (F[1,15] = 0.6269, p = 0.441), and total distance ANOVA revealed no signiﬁcant surgery group effect (F[1,22] = (F[1,15] = 0.1784, p = 0.679) in the open ﬁeld test (Supplemen- 0.0123, p = 0.9126) or deprivation level × surgery group interac- tary Fig. 1b). Thus, observed contextual episodic and spatial tion (F[1,22] = 0.1665, p = 0.687). Animals in Group 0 + working memory impairments observed in SDV and CCK-SAP responded signiﬁcantly more under 0 h than under 24 h food rats are unlikely to be secondary to effects on anxiety-like beha- deprivation (Fig. 2d, left) and those in Group 24+ responded vior. Overall, there were three cohorts of SDV and sham signiﬁcantly more under 24 h than 0 h food deprivation (Fig. 2d, animals, and terminal body weights did not differ between sur- right), regardless of surgical group. These conclusions are gical groups in any cohort: cohort 1 underwent deprivation Fig. 2 SDV and CCK-Sap impair HPC-dependent contextual episodic and spatial working memory, but not interoceptive, social, or olfactory learning. a SDV (n = 6) impairs contextual episodic memory relative to controls (n = 9); discrimination index on day 1 (habituation) and day 3 (test day) of NOIC testing (repeated-measures ANOVA, F[1,13]= 5.564, p = 0.0347; Newman–Keuls’ post hoc, p = 0.0047). b SDV (n = 8) impairs spatial working memory relative to controls (n = 8); difference in number of errors from trial 2 (T2) to trial 1 (T1) across individual training days (left) (ANOVA, F[1,14]= 3.626, p = 0.0776 (Day 2), F[1,14]= 3.842, p = 0.0702 (Day 4), F[1,14]= 3.555, p = 0.0803 (Day 5)) and the average T2–T1 errors for each training day in the Barnes maze test (repeated-measures ANOVA, F[1,19] = 6.8565, p = 0.0169). c, d SDV does not impact deprivation intensity discrimination performance; c pre-surgery training (Group 0+ , n = 16; Group 24 + , n = 11; repeated-measures ANOVA, F[1,22]= 135.54, p < 0.0001; Newman–Keuls’ post hoc, Group 0 + block 3–6 all p < 0.0017, Group 24+ block 2–6 all p < 0.000178), d and post-surgery testing [mean percent of 20 s epochs of interval magazine entries during the last minute of test session for Group 0 + (sham, n = 8; SDV, n = 7) and 24 + (sham, n = 6; SDV, n = 5) under alternating 0 h and 24 h food restriction] (repeated-measures ANOVA, F[1,22] = 80.5115, p < 0.00001; Newman–Keuls’ post hoc, all p < 0.004). e SDV (n = 6) does not impact STFP relative to controls (n= 9); 30 min percent preference for the socially paired ﬂavored chow and 30 min cumulative food intake (grams) in the STFP test (paired t-test, p = 0.014 (SDV), p = 0.014 (sham)). f SDV does not impact anxiety-like behavior; time spent in open arm section (seconds) and number of open section entries during zero maze test for the SDV vs. sham groups. g CCK-SAP impairs contextual episodic memory; NOIC discrimination index on days 1 and 3 in CCK-SAP (n = 9) and SAP (n = 8) control rats (repeated-measures ANOVA, F[1,15] = 6.496, p = 0.0223; Newman–Keuls’ post hoc, p = 0.0241). h CCK- SAP impairs SWM; (T2–T1 error for each individual training day (ANOVA, F[1,13]= 6.824, p = 0.0215 (Day 2)) and overall average (repeated-measures ANOVA, F[1,13] = 8.66, p = 0.0114) in CCK-SAP (n = 8) and SAP (n = 7) control rats. (*P < 0.05; P < 0.08 vs. sham or SAP controls; data are mean ± SEM) 4 NATURE COMMUNICATIONS (2018) 9:2181 DOI: 10.1038/s41467-018-04639-1 www.nature.com/naturecommunications | | | NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04639-1 ARTICLE intensity discrimination task (sham: 413.54 ± 10.74, SDV: 396.78 ANOVA) (Fig. 4b, d) relative to i.p. saline treatment. In addition, ± 11.62; p = 0.329, ANOVA), cohort 2 tested in the Barnes task i.p. CCK injections increased the number of labeled cells for c-Fos (sham: 391.19 ± 8.33, SDV: 365.09 ± 11.95; p = 0.095, ANOVA), messenger RNA (ﬂuorescent in situ hybridization, FISH) and cohort 3 tested in NOIC and STFP tasks (sham: 371.69 ± expressed in the dCA3 (F[1,9] = 53.093, p = 0.000046, ANOVA) 7.16, SDV: 354.47 ± 11.42; p = 0.199, ANOVA). Similarly, the (Fig. 5a) and DG (F[1,9] = 40.496, p = 0.000131, ANOVA) cohort of SAP and CCK-SAP animals used in this study showed (Fig. 5b) relative to saline treatment, with 93.11% and 94.35% of no signiﬁcant group differences in terminal body weight (SAP: c-Fos mRNA-positive cells in dCA3 (Fig. 5c) and DG (Fig. 5d) 394.16 ± 11.04, CCK-SAP: 383.28 ± 11.53; p = 0.508, ANOVA). being VGLUT1 positive, respectively, and only 4.29% (Figs. 5e) Therefore, we conclude that the observed contextual episodic and and 8.44% (Fig. 5f) of c-Fos mRNA-positive cells being GAD2 spatial working memory impairments observed in SDV and mRNA positive, respectively. CCK-SAP rats are unlikely to be secondary to surgical effects on body weight regulation. Medial septum connects mNTS neurons to the dorsal HPC. The medial nucleus tractus solitarius (mNTS) is the ﬁrst central SDV and CCK-SAP reduce BDNF and DCX in the dHP. nervous system (CNS) site to receive GI-derived vagal sensory Immunoblot analyses from dorsal HPC lysates revealed that total input; however, the mNTS does not communicate mono- SDV reduced BDNF and DCX protein expression in the dorsal synaptically with the HPC . To identify regions of possible relay HPC relative to sham controls (Fig. 3a, b), with a signiﬁcant main between the mNTS and HPC, a combination of retrograde and effect of surgical group observed for BDNF (F[1,14] = 4.609, p = anterograde pathway tracing was used: unilateral iontophoretic 0.049, ANOVA) and DCX (F[1,14] = 5.5133, p = 0.034, injections of a retrograde pathway tracer targeted to the dCA3 ANOVA). Similar to SDV, the CCK-SAP signiﬁcantly reduced (cholera toxin subunit B (CTB) AlexaFluor 488 (AF488) con- levels of both proteins in the dorsal HPC relative to SAP controls jugated) (Fig. 6a, c) were combined with ipsilateral iontophoretic (Fig. 3c, d). This conclusion is supported by one-way ANOVA injections of an anterograde pathway tracer targeted to the mNTS analyses indicating a signiﬁcant main effect of surgical group for (AAV1-hSyn-TurboRFP-WPRE-rBG) (Fig. 6b, d). Red ﬂuor- both dorsal hippocampal BDNF (F[1,13] = 4.881, p = 0.0457) ophore (red ﬂuorescent protein (RFP) and Cy3 following and DCX (F [1,14] = 5.494, p = 0.034) levels. On the other hand, immunohistochemistry (IHC)) anterogradely labeled axons ori- immunoblot analyses from whole hypothalamic lysates revealed ginating from the mNTS were found in apposition to green no signiﬁcant group differences for both BDNF (F[1,13] = 0.26, ﬂuorophore (AF488 following IHC) labeled cell bodies in the MS p = 0.619, ANOVA) and DCX (F[1,14] = 0.4955, p = 0.493, that were retrogradely labeled from dCA3 in each of the three ANOVA) levels in CCK-SAP vs. control SAP animals (Supple- animals that were conﬁrmed as double hits in both injection sites mentary Fig. 2 a, b), indicating that GI vagal afferent ablation is (representative appositions in Fig. 6e–g), whereas such apposi- unlikely to have systemic brain-wide impact of these neuro- tions were not observed in various control animals (n = 11) in trophic (BDNF) and neurogenic (DCX) markers. Hypothalamic which either (or both) injection site(s) were either undetermin- tissue was not collected in SDV and sham groups, and therefore able or adjacent to the intended target. analyses for these groups could not be included. To further support this multi-order pathway, we utilized a Following Shapiro–Wilk test of normality to conﬁrm that the novel dual-synaptic virus-based pathway tracing approach to data were normally distributed [CCK-SAP vs. SAP controls: examine whether mNTS neurons synpatically communicate to BDNF, W = 0.75377; DCX, W = 0.90026; DI, W = 0.96494; the HPC via a MS relay pathway . The AAV2/1-hSyn-Cre drives spatial working memory, W = 0.94624; for SDV vs. sham Cre expression in ﬁrst-order neurons infected at the injection site, controls: BDNF, W = 0.88919; DCX, W = 0.92599; DI, W = as well as in second-order (but not third-order) neurons based on 0.93305; spatial working memory, W = 0.91989], linear regres- virion release from ﬁrst-order axon terminals . A unilateral sion analyses were conducted to examine whether dorsal iontophoretic co-injection of AAV2/1-hSyn-Cre (and CTB-488 to hippocampal BDNF and DCX protein expression were function- conﬁrm injection site) targeted to the mNTS (at the level of the ally correlated with HPC-dependent spatial working memory area postrema; Fig. 7a) was followed by a pressure injection of (Fig. 3e, f) and NOIC (Fig. 3g, h) task performance. For Barnes AAV1-CAG-FLEX-TdTomato (a Cre-dependent anterograde maze, analyses included all groups (sham, SDV, SAP, and CCK- tracer) targeted to the MS (Fig. 7b, c). Results revealed robust SAP) and revealed that (1) dorsal HPC BDNF levels are axon terminal ﬁelds in the dCA3 (Fig. 7d, e) and DG (Fig. 7h, i) negatively correlated with average error difference (T2–T1) (F for each of the three animals that were conﬁrmed as double hits [1,28] = 4.211, R2 = 0.1307, p = 0.0496) and (2) dorsal HPC in both injection sites. A schematic of the rostral-caudal DCX levels showed a trend toward signiﬁcant correlation with distribution of axon terminal ﬁelds in the dCA3 (Fig. 7f, g) and Barnes performance (F[1,29] = 3.546, R2 = 0.1089, p = 0.0698). DG (Fig. 7j, k) from a representative double hit animal are These results indicate that lower levels of BDNF and DCX are displayed. Axonal labeling in the dHPC was not observed for rats associated with poorer performance in the Barnes task. For (n = 13) in which either the mNTS or MS injection was absent or NOIC, analyses of SAP and CCK-SAP groups only (as HPC adjacent to the targeted region. Overall, these results indicate the lysates were not collected from SDV and sham rats that presence of synaptic connections to the dHPC from MS neurons performed NOIC) reveal a signiﬁcant positive correlation for that receive direct input from the mNTS. both BDNF (F[1,13] = 5.277, R2 = 0.2887, p = 0.0389) and DCX (F[1,13] = 7.36, R2 = 0.3615, p = 0.0178) levels with DI, indicat- Discussion ing that lower levels of BDNF and DCX are associated with Our results reveal that GI-derived vagal sensory signaling endo- poorer performance in contextual episodic memory. genously promotes hippocampal-dependent learning and mem- ory function in rats. Both classic nonselective (SDV; eliminates all CCK activates c-Fos in dCA3 and DG glutamatergic neurons. GI vagal afferents and efferents) and novel sensory-selective Intraperitoneal (i.p.) injections of CCK-8 (8 μg/kg) increased the (CCK-SAP approach; eliminates ~ 80% of upper GI vagal affer- number of c-Fos protein immunoreactive cells expressed in the ents) ablative methods of GI vagal disconnection impaired hip- dorsal CA3 (dCA3; F[1,9] = 20.236, p = 0.001492, ANOVA) pocampal (HPC)-dependent memory processes, including spatial (Fig. 4a, c) and dentate gyrus (DG; F[1,9] = 37.917, p = 0.000167, working memory and contextual episodic memory. Moreover, NATURE COMMUNICATIONS (2018) 9:2181 DOI: 10.1038/s41467-018-04639-1 www.nature.com/naturecommunications 5 | | | ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04639-1 both ablative methods reduced expression of neurogenic (DCX) in glutamatergic neurons in the dorsal HPC CA3 and DG. To and neurotrophic (BDNF) markers in the dorsal HPC that pro- identify multi-order neuronal pathways by which GI vagal sen- mote neurogenesis and plasticity, and expression of these markers sory signaling communicates to the HPC, we employed multiple in the HPC were correlated to both spatial working memory and innovative multi-order neural pathway tracing strategies. These contextual episodic memory. We further investigated HPC approaches identiﬁed the MS as a relay region between the mNTS involvement in GI vagal sensory signaling by analyzing neuronal (ﬁrst site of vagal sensory input to the CNS) and the dorsal HPC activation in the HPC in response to the GI-derived vagally (CA3 and DG). Overall, these results reveal a novel role for gut- mediated satiation signal, CCK. Expression of c-Fos in response to-brain communication in the control of learning and memory to peripheral CCK was robust in the HPC, predominantly present ab Sham SDV Sham SDV Sham SDV Sham SDV BDNF 14 kDa DCX 45 kDa β-Actin 42 kDa β-Tubulin 50 kDa Dorsal hippocampus BDNF protein expression Dorsal hippocampus DCX protein expression 1.4 Sham SDV 1.4 Sham SDV 1.2 1.2 0.8 * 0.8 0.6 * 0.6 0.4 0.4 0.2 0.2 cd Sap CCK-Sap Sap CCK-Sap Sap CCK-Sap Sap CCK-Sap 14 kDa BDNF DCX 45 kDa β-Actin 42 kDa β-Tubulin 50 kDa Dorsal hippocampus DCX protein expression Dorsal hippocampus BDNF protein expression 1.4 1.4 Saporin CCK-Saporin Saporin CCK-Saporin 1.2 1.2 0.8 0.8 * 0.6 0.6 0.4 0.4 0.2 0.2 ef y = –3.939x + 1.096 y = –2.886x – 0.2798 Sham Sham SDV 2 SDV 2 R = 0.1307 R = 0.1089 Sap Sap CCK-Sap P -val = 0.0496* CCK-Sap P -val = 0.0698 5 5 0.5 1.0 1.5 2.0 0.5 1.0 1.5 2.0 –5 –5 –10 –10 –15 –15 BDNF DCX gh 1.0 1.0 Sap Sap CCK-Sap CCK-Sap 0.8 0.8 0.6 0.6 0.4 0.4 y = 0.1021x + 0.513 y = 0.1147x + 0.4906 0.2 2 0.2 R = 0.2887 2 R = 0.3615 P -val = 0.0389* P -val = 0.0178* 0.0 0.0 0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0 BDNF DCX 6 NATURE COMMUNICATIONS (2018) 9:2181 DOI: 10.1038/s41467-018-04639-1 www.nature.com/naturecommunications | | | Average errors T2-T1 Discrimination index Relative BDNF/ β-Actin Relative BDNF/ β-Actin Average errors T2-T1 Discrimination index Relative DCX/ β-tubulin Relative DCX/ β-Tubulin NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04639-1 ARTICLE function and identify a putative neuronal pathway through which improved cognition following 6 months of chronic cervical vagus this communication may occur. nerve stimulation treatment . At a mechanistic level, recent Previous work using electrical vagus nerve stimulation studies have indicated a functional role for the gut microbiome in approaches involving non-physiological stimulation of the cer- regulating the interaction between GI signaling and cognition . vical vagus nerve (and is therefore not selective to GI vagal sig- For example, in germ-free mice that lack a microbiome, hippo- naling) revealed improved word-recognition memory in humans campal BDNF levels are reduced , and these effects are asso- 8 25 following vagus nerve stimulation . Similarly in rodents, vagus ciated with cognitive dysfunction . In addition, total SDV nerve stimulation improved retention of inhibitory-avoidance impairs the ability of butyrate, a short-chain fatty acid synthesized 7 19 memory and facilitated extinction of conditioned fear . Our by colonic microbiota, to reduce appetite and prevent diet- data expand these ﬁndings by establishing a physiological role for induced obesity , suggesting a critical need for preservation of vagal sensory signaling, speciﬁcally that originating in the GI vagal nerve signaling in the microbiome–gut–brain axis. Given tract, in HPC-dependent memory function. These ﬁndings may that butyrate promotes hippocampal neurogenesis and memory have clinical relevance in relation to current treatments for obe- function , one possible mechanism linking GI vagal afferent sity that involve disruptive manipulation of the vagus nerve, such signaling to HPC function is via gut microbial interactions with as bariatric surgeries (e.g., RYGB, vertical sleeve gastrectomy) short-chain fatty acid production. and chronic electrical disruption of vagal nerve signaling (e.g., We examined the effects of SDV-mediated GI vagal ablative VBLOC ). disconnection on a variety of HPC-dependent mnemonic and Our results support the notion that gut to brain vagally- cognitive processes that rely on the utilization of different cues mediated communication has an important role in protecting (external, interoceptive, social, olfactory), and that differ in their against neurodegenerative disorders (e.g., Alzheimer’s disease). reliance on discrete HPC subregions. Of the various memory For example, human patients with Alzheimer’s disease showed procedures assessed, SDV signiﬁcantly impaired spatial working ab Dorsal CA3 (dCA3) cFos protein expression Dentate gyrus (DG) cFos protein expression 14 14 i.p. saline i.p. CCK i.p. saline i.p. CCK 12 12 10 10 8 8 6 6 2 2 0 0 cd dCA3 DG DGmo CA3sr CA3sr DGmo DGsg DGpo DGsg DGpo CA3sp CA3sp DGmo DGmo i.p. saline i.p. CCK i.p. saline i.p. CCK Fig. 4 Peripheral administration of CCK activates c-Fos protein expression in the dorsal CA3 (dCA3) and dentate gyrus (DG). Intraperitoneal injections of CCK (n = 6) (a vagally mediated gastrointestinal-derived satiation signal) increases the number of c-Fos-immunoreactive (-ir) cells (a marker for neural activation) expressed in the a dCA3 and b DG vs. saline (n = 5) treatment (ANOVA, F[1,9]= 20.236, p = 0.001492 (dCA3), F[1,9]= 37.917, p = 0.000167 (DG)). Representative images of immunohistochemical staining of c-Fos-ir protein (green) in the c dCA3 and d DG. Scale bar: 25 μm. (*P < 0.05 vs. i.p. saline controls; data are mean ± SEM.; CCK cholesystokinin, i.p. intraperional, DG dentate gyrus, dCA3 dorsal CA3, CA3sr CA3 stratum radiatum, CA3sp CA3 pyramidal layer, DGmo dentate gyrus molecular layer, DGpo DG polymorph layer, DGsg DG granule cell layer) Fig. 3 SDV and CCK-SAP reduce BDNF and DCX protein expression in the dorsal HPC and are functionally related to HPC-dependent memory performance. a, b SDV reduces protein expression of BDNF and DCX (expressed relative to loading control proteins) in dorsal HPC tissue in SDV (n = 8) vs. sham-operated control rats (n = 8) (ANOVA, F[1,14]= 4.609, p = 0.049 (BDNF), F[1,14]= 5.5133, p = 0.034 (DCX)). c, d CCK-SAP-mediated GI vagal afferent ablation (n = 8) reduces dorsal HPC BDNF and DCX expression relative to SAP (SAP BDNF, n = 7; SAP DCX, n = 8) controls (ANOVA, F [1,13]= 4.881, p= 0.0457 (BDNF), F[1,14]= 5.494, p= 0.034 (DCX). e–h Linear regression of average number of errors from trial 2 to trial 1 (spatial working memory) and NOIC discrimination index (contextual episodic memory) against relative BDNF and DCX expression reveals a signiﬁcant negative correlation for BDNF (F[1,28]= 4.211, R2 = 0.1307, p = 0.0496) (e) with a trend for DCX (F[1,29] = 3.546, R2 = 0.1089, p = 0.0698) (f). For the novel object in context (NOIC) task of contextual episodic memory, there was a positive correlation between discrimination index and protein expression of BDNF (F[1,13]= 5.277, R2 = 0.2887, p = 0.0389) (g) and DCX (F[1,13] = 7.36, R2 = 0.3615, p = 0.0178) (h). (*P < 0.05 vs. controls [sham and/or SAP]; data are mean ± SEM. BDNF brain-derived neurotrophic factor, CCK-SAP cholecystokinin–saporin, DCX doublecortin, SDV subdiaphragmatic vagotomy NATURE COMMUNICATIONS (2018) 9:2181 DOI: 10.1038/s41467-018-04639-1 www.nature.com/naturecommunications 7 | | | # of cFos+ cells # of cFos+ cells ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04639-1 ab Dorsal CA3 (dCA3) cFos mRNA expression Dentate gyrus (DG) cFos mRNA expression i.p. saline i.p. CCK i.p. saline i.p. CCK 16 16 12 12 10 10 8 8 6 6 4 4 0 0 –2 –2 c d cFos VGLUT1 cFos VGLUT1 DAPI dCA3 DAPI DG ef cFos GAD2 cFos GAD2 DAPI dCA3 DAPI DG Fig. 5 Peripheral administration of CCK activates c-Fos mRNA expression in dCA3 and DG hippocampal glutamatergic neurons. Number of c-Fos-labeled (c-Fos+ ) cells for mRNA (ﬂuorescent in situ hybridization) expressed in the a dCA3 and b DG following i.p. administration of CCK (n = 6) or saline (n = 5) (ANOVA, F[1,9] = 53.093, p = 0.000046 (dCA3), F[1,9] = 40.496, p= 0.000131 (DG)). Approximately 93% and 94% of c-Fos+ cells in the dCA3 and DG were VGLUT1+ (respectively) following i.p. CCK treatment, whereas only 4% and 8% of c-Fos + cells in the dCA3 and DG were GAD2 + (respectively) following i.p. CCK. c, d Representative images show c-Fos mRNA (green) and VGLUT1 (red) or GAD2 (e, f) (red) mRNA expression in dCA3 (c, e) and DG (d, f) cell bodies following i.p. CCK (DAPI nuclear stain; blue). Scale bar: 25 μm. (Arrows, co-expression of c-Fos/VGLUT1 mRNA cells; *P < 0.05 vs i.p. saline controls; data are mean ± SEM. CCK cholesystokinin, dCA3 dorsal CA3, DG dentate gyrus, i.p. intraperional) memory (Barnes maze procedure) and contextual episodic predominantly with visuospatial-based exteroceptive memory, memory (NOIC), whereas performance in appetitive learning whereas the ventral (temporal in rodents, anterior in primates) based on internal energy-state cues (deprivation intensity dis- HPC is associated with conditioned appetitive and anxiety-like 2, 28, 29 crimination) and social transmission of food-related (olfactory) behaviors . The contextual episodic (NOIC) and spatial cues (STFP) was preserved. The lack of group differences between working memory (Barnes maze) tasks used in the present study SDV and control animals for both deprivation intensity dis- rely on the integration of visuospatial external environmental crimination and STFP learning could be due to differential effects cues. Conversely, deprivation intensity discrimination and STFP of vagal nerve signaling on two functionally and anatomically- rely on conditioned appetitive cues (internal energy-state and independent subregions of the HPC, as studies have shown the social cues, respectively). Deprivation intensity discrimination dorsal (septal in rodents, posterior in primates) HPC is associated learning is impaired by lesions to complete, dorsal, or ventral 8 NATURE COMMUNICATIONS (2018) 9:2181 DOI: 10.1038/s41467-018-04639-1 www.nature.com/naturecommunications | | | # of cFos+ mRNA cells #of cFos+ mRNA cells och V3p 2 isl isl is l MS SH LSc lot MOp ul i s l OT r.dl.m.d FS NDB C P r.dl.l.d c.v.l ac o S I AC B SSp r.vl.d.m r.m.d r.vl.d.l r.m.v.r AUDv RT SSs CL chpl r.m.v.r r.vl.d.l fi r.m.d r.vl.d.m SSp PERI sptV alv CEAl AC B MEApv bfd SI ac o SPVC c.v.l PARN COApl r.dl.l.d CA2 CP NDB SI FS r.dl.m.d CP BMAp IOma PAA IA OT cuf GPe py alv SSp CM CU PAT tr sm hf NTSl isl ts ml mo ECU NTSm ul MOp EPv sg lot FC LSc AMBd AMBv rust MS isl 2 isl SH is l V3p och NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04639-1 ARTICLE ab GR AP DMX po VIP XII IMD PMR PH 5 b 1 DMHa 4 3 in ex L29 L70 cd AP DG CA3 mNTS c.d.r L15 ef g Fig. 6 Co-injection monosynaptic neural pathway tracing strategy identiﬁes the medial septum (MS) as a relay region connecting the mNTS to the dHPC (CA3). Schematic representative injection sites in a dCA3 and b mNTS in Swanson Atlas level 29 and 69, respectively. c Unilateral iontophoretic dCA3 injection site of the retrograde tracer, CTB-488 (green). Scale bar: 100 μm. d Ipsilateral and unilateral iontophoretic mNTS delivery of the anterograde viral tracer, AAV1-TurboRFP (red) (n = 3 double hits, n= 11 controls). Scale bar: 500 μm. e, f, g RFP-ir axons from the mNTS in apposition to CTB-488-ir cell bodies from dCA3 in the MS (images made by authors and adapted from Swanson Atlas level 15 ). Scale bar: 10 μm. (AP area postrema, DG dentate gyrus, mNTS medial nucleus tractus solitarius) NATURE COMMUNICATIONS (2018) 9:2181 DOI: 10.1038/s41467-018-04639-1 www.nature.com/naturecommunications 9 | | | 6a ec df VL V3t LD MH LH PVT MDc l RH SMT cd RE PR cp mt V3r LHA V3h fx st PVi 3 3 ARH st ME TUsv 1 3 CA3 mlf MDRNv VL LSr 6a 6b rf LPO tsp LHAd LHAs LHAjd IG IG IA MOp CA1 Z I VM ccg st cc e m DGlb SE Z spd IA cing slu MOs VMHc LHAjvd dm vl slm PIR sg mo DGmb sctv LRNm opt PCN so cpd sup sps LHAm sr sfp vm ec ECT LRNp vlt MEApd MOs LHAjvv VPM ec TUi TUl VPL VA L EPd AIp co PI R GU BLAa CLA RSPd sctd EPd ci ng LA VISC amc BLAp int TEa SSs ACAd ACAv RSPv ACAv ENTl ACAd SSs VISC cing CLA GU AIp EPd ec MOs P IR SE Z ccg IG rf LPO 6b 6a VL LSr c.d.r n chpl fi alv ec alv FC CA2 DGcr CA2 alv hf sg sm sm CA3 mo FC ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04639-1 6, 29 HPC , whereas STFP is primarily linked with ventral hippo- Considering these ﬁndings, we hypothesize that interoceptive campal substrates . Thus, overall our data suggest gut-derived processing for deprivation intensity discrimination learning is vagal signaling promotes memory involving external environ- sustained in the absence of GI vagal input, and may therefore mental cues (that may rely predominantly on the dorsal HPC), primarily involve circulating endocrine and other metabolic sig- while having less impact on conditioned appetitive and anxiety- nals that communicate to the HPC. Consistent with this frame- like behaviors (that may rely predominantly on the ventral HPC). work, injections of the gut-derived hunger hormone, ghrelin, into ab c MS AP mNTS aco cc d e CA3so CA3sr CA3sp L29 slu L28 L30 h i DGmo DGpo DGsg L29 j k VIP chf3 mo VIP L28 L30 10 NATURE COMMUNICATIONS (2018) 9:2181 DOI: 10.1038/s41467-018-04639-1 www.nature.com/naturecommunications | | | 6a 6a 6b VL LD df df V3t V3t MH LH MH LH po alv CA1 CA3 sg sg cc po slm sr po mo sg DGlb DGlb DGmb hf DGmb slu so DGlb DGlb sp ing DGmb DGmb CA1 sg mo slm sg sr so sps cing NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04639-1 ARTICLE the ventral but not dorsal HPC increase food intake , and the animal must remember, which object it has previously seen), intracerebroventricular ghrelin injections generalize to 24 h food the NOIC test relies on external visuospatial and/or contextual restriction in non-restricted rats trained in the deprivation dis- memory (i.e., animal must remember the location in which it crimination task . However, one limitation of the design with previously encountered an object). Based on previous work, it regards to comparing across these different tasks is that the remains controversial whether the HPC is critically involved in learning of interoceptive and social cue-based tasks occurred at novel object recognition learning, but rather mediates recognition different times post surgery. Further, although the experimental memory when it requires remembering that a stimulus occurred design was consistent across surgical groups with regards to time in a certain place or time . The perirhinal cortical area, on the between surgery and behavioral testing, complete counter- other hand, is more strongly linked novel object recognition balancing of behavioral experiments that involved multiple memory, as lesions to this brain region impair the ability of 37–39 comparisons was not employed and is therefore a limitation of animals to discriminate between familiar and novel objects . the study. Moreover, rats presented with novel versus familiar objects or Episodic memory, or memory of a speciﬁc event, is HPC- pictures have increased c-Fos expression in perirhinal cortex but dependent and was impaired by SDV (NOIC procedure). Epi- not HPC, whereas the HPC, but not perirhinal cortex shows sodic memories are important for the control of feeding behavior increased c-Fos expression in response to novel spatial arrange- 40, 41 and energy balance, as they are critical for animals to remember ments of familiar objects . Whether or not the SDA approach, aspects about where (food location), what (nutritive vs. adverse like SDV and CCK-SAP approaches, impairs spatial and/or postingestive consequences), and when eating occurs. Consistent contextual-based HPC-dependent memory requires further study. with this notion, experimental manipulations in human subjects STFP and NOIC involve consumption of novel foods and designed to disrupt episodic memory during feeding increase exposure to novel objects, respectively, which are also used to hunger ratings and food intake at a subsequent eating assess anxiety-like behavior in rodents (neophobia). Thus, we 32, 33 episode . Similarly in rats, disruption of episodic meal-related tested both SDV and CCK-SAP rats in the zero maze anxiety test. memory via postprandial dorsal hippocampal infusion of mus- Moreover, we also tested the CCK-SAP animals in an additional cimol decreases the latency to start the post-infusion meal and anxiety-relevant task, the open ﬁeld test. Results revealed no increases the size of the post-infusion meal . From an evolu- signiﬁcant group differences in either SDV or CCK-SAP groups tionary perspective, the physiological role of GI-derived vagal relative to controls in these anxiety-related tests. These results sensory signaling in HPC-dependent memory may normally differ from a previous study that demonstrated reduced innate function to enhance episodic memory for eating occasions, as GI anxiety-like behavior in SDA rats . These effects could be due to vagal sensory signaling is most heavily engaged during feeding. procedural differences, as unlike the individually housed rats in Moreover, given that it is advantageous to remember the physical the present study, the rats in the previous study were group- location of the food source to inform future foraging behavior, the housed, which has been shown to demonstrate a decrease in visuospatial external environment is likely to be a critical com- mean arterial blood pressure and heart rate relative to isolated 43, 44 ponent of episodic meal-related memories. From this perspective, (single-housed) male and female Sprague–Dawley rats .In GI vagal sensory signaling during meal taking represents an addition, we tested the SDV and CCK-SAP groups in an elevated advantageous biological survival mechanism that promotes meal- zero maze, whereas the SDA rats in the previous study were tested related episodic memory to facilitate future feeding. in an elevated plus maze. Although both the zero maze and the Based on the impaired spatial working and contextual episodic elevated plus maze are well accepted tests for measuring anxiety- like behavior in rodents, untreated/normal rats show increased memory following SDV (that eliminates both GI sensory and motor signaling), we next demonstrated that a novel selective GI exploration time of the open areas in the elevated plus vs. zero vagal sensory ablation surgical method (CCK-SAP) also impaired maze, potentially due to the time spent in the center (neutral) both of these HPC-dependent memory processes. These ﬁndings region of the plus maze, to which these is no equivalent in the 45, 46 suggest that GI vagal sensory/afferent (and not motor/efferent) zero maze . Although a more extensive analysis of anxiety was signaling promotes HPC-dependent memory. The CCK-SAP conducted in this previous study (we did not perform the food approach selectively eliminates ~ 80% of GI-derived vagal afferent neophagia test), it is worth noting that the elimination of 50% of signaling below the diaphragm , which differs from the estab- the vagal afferents above the diaphragm via SDA could be a lished surgical subdiaphragmatic deafferentation procedure potential reason for different innate anxiety-like effects of SDA (SDA), which involves cutting the vagal sensory (afferent) rootlets relative to SDV and CCK-SAP animals, as the supradiaphramatic unilaterally near the brainstem interface and then ablating the vagal afferents are preserved in these two latter approaches. contralateral subdiaphragmatic vagal trunk. SDA eliminates all GI Consistent with this possibility, optogenetic activation of non- vagal to mNTS sensory signaling, while leaving 50% of supra- selective vagal afferents (including those innervating cardiac diaphragmatic sensory and 50% of vagal motor (efferent) sig- systems) robustly reduces heart rate in mice , and transgenic naling intact . Previous work has shown that SDA has no effect overexpression of angiotensin-(1–7) in mice chronically reduces on novel object recognition memory or working memory in a heart rate and is accompanied by reduced anxiety-like behavior . non-spatial alternation task . In contrast, here we show that both Future research is needed to directly examine the role of different SDV and CCK-SAP impairs a similar NOIC task. Although novel vagal afferent neuron populations in anxiety-like behavior. object recognition relies on visual object recognition memory (i.e., Fig. 7 Multisynaptic viral tracing approach reveals MS neurons that receive monosynaptic input from the mNTS directly project to the dHPC (dCA3 and DG). a Unilateral iontophoretic co-injection of AAV2/1-hSyn-Cre and CTB (CTB-ir in green; to conﬁrm injection site placement) in the mNTS (n = 3 double hits, n = 13 controls), which drives Cre expression in second-order (but not third-order) neurons based on synaptic virion release from ﬁrst-order axon terminals . Scale bar: 100 μm. b, c A 200 nl pressure injection site of a Cre-dependent anterograde tracer (AAV1-CAG-FLEX-TdTomato) in the MS Scale bar: b 200 μm, c 50 μm. Axon terminal ﬁelds in the d, e dCA3 and h, i DG of MS neurons that receive direct input from mNTS. Scale bar: d, h 250 μm, e, i 50 μm. A schematic representation of dCA3 (f, g) and DG (j, k) axon terminal ﬁeld distribution (Made by co-author and adapted from Swanson atlas level 28–30 ). (aco anterior commissure, AP area postrema, CA3sr CA3 stratum radiatum, CA3sp CA3 pyramidal layer, DGmo dentate gyrus molecular layer, DGpo DG polymorph layer, DGsg DG granule cell layer, mNTS medial nucleus tractus solitarius, MS, medial septum) NATURE COMMUNICATIONS (2018) 9:2181 DOI: 10.1038/s41467-018-04639-1 www.nature.com/naturecommunications 11 | | | ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04639-1 The mNTS, where GI vagal sensory input arrives in the CNS, (treatments given counterbalanced on separate days) after an overnight fast. SDV rats were included in the statistical analysis if CCK treatment resulted in a < 30% does not send direct projections to the HPC . Here we show that 57, 58 reduction of their food intake, as described . Of the three separate cohorts of peripheral injections of CCK, a vagally mediated GI-derived rats that underwent SDV surgery and subsequent behavioral testing, four animals satiation signal, signiﬁcantly increased c-Fos protein and mRNA were removed from deprivation intensity discrimination analyses and one animal expression in the dorsal HPC CA3 and DG, suggesting a multi- was removed from NOIC analyses based on these criteria. order connection between mNTS and the dHPC. We utilized a monosynaptic co-injection neural pathway tracing method to CCK-SAP nodose ganglia injection. CCK-SAP targets CCK receptor expressing identify the MS as a possible relay region connecting mNTS to the cells, which are localized on vagal afferent neurons that speciﬁcally innervate the 59, 60 upper GI tract . As recently conﬁrmed, CCK-SAP injection in the nodose HPC . As this method does not determine synaptic commu- ganglia (vagal afferent cell bodies) selectively eliminates ~ 80% of GI-derived vagal nication between immunoreactive cell bodies (back-labeled from afferent signaling, while preserving colonic and supradiaphragmatic vagal sensory the CA3) in apposition to axon terminals emanating from the pathways, as well as all vagal efferents . Twenty-four hours before surgery, rats mNTS, we employed a dual-synaptic viral-mediated anterograde were given 15 ml of condensed milk in addition to their normal ad libitum access to chow and water, and were fasted before lights went off (18:00 h). Twenty minutes tracing method and conﬁrmed the MS as a mNTS to HPC relay before surgery, rats received an i.p. injection of atropine sulfate (0.05 mg/kg) and region . Although the dual-synaptic AAV1-Cre can be trans- carprofen (5.0 mg/kg; Henry Schein), and then anesthetized with a ketamine (90 ported in both the anterograde and retrograde (from axon mg/kg), xylazine (2.7 mg/kg), and acepromazine (0.64 mg/kg) cocktail. A midline terminals) direction , we limited the application to a pathway incision was made along the length of the neck. The vagus nerve was separated where there are no reciprocal connections between targeted pre- from the carotid artery with Graefe forceps until the nodose ganglion was visible and accessible. A glass capillary (20 μm tip, beveled 30° angle) attached to a and postsynaptic regions. While the mNTS projects to the MS, micromanipulator was used to position and puncture the nodose ganglion and 1 µl descending MS projections do not project to the caudal brain- volume of CCK-SAP (250 ng/µl) or SAP (250 ng/µl) was injected with a Picos- stem . Previous studies have established a role of septal choli- pritzer III injector (Parker Hanniﬁn) at two sites: 0.5 µl rostral and 0.5 µl caudal to nergic function in HPC-dependent learning and memory the laryngeal nerve branch. The same procedure was performed for both nodose ganglia on either side before the skin was closed with sterile and stop sutures along processes. Future work could investigate whether cholinergic the skin. Rats were returned to their home cage and deprived of water for 6 h and signaling is critical in regulating GI vagal modulation of HPC- food overnight. The post-op care was optimized to avoid excessive weight loss post dependent spatial working and episodic memory function. 61 surgery and increase survival rate, as previously described . The schedule was as Collective results from the present study demonstrate that follows: Day 1 post-op rats received carprofen (5.0 mg/kg; SQ) and were given ad libitum access to condensed milk; day 2 post-op rats received mash (10 g powdered endogenous vagal afferent signaling from the GI tract regulates chow mixed with 20 ml condensed milk diluted as described above); day 3 post-op HPC-dependent contextual episodic and spatial working mem- rats received mash and solid chow pellets; day 4 and onwards, rats were given ad ory, potentially by driving the expression of memory-related libitum access to chow. After behavioral testing, CCK-SAP was veriﬁed functionally neurotrophic (BDNF) and neurogenic (DCX) signaling pathways. with i.p. CCK-induced food intake reduction. The functional veriﬁcation modeled that of the SDV approach described above and that published for CCK-SAP in . These ﬁndings compliment and expand previous work using Based on this veriﬁcation test, four CCK-SAP rats were removed from all analyses. cervical vagus nerve stimulation, as well as a recent report showing that total SDV in mice reduces HPC DCX . We further identify the MS as a likely relay connecting the gut to glutama- General research design. Three separate cohorts of rats underwent SDV (or sham) surgery and subsequent behavioral testing (described below), beginning tergic dorsal HPC neurons. Our results further expand previous 7 days post surgery. Cohort 1 underwent deprivation intensity discrimination 36, 42 work by revealing that gut-derived signals, either vagal or training (described below) in which rats were assigned to one of two groups (group endocrine , interact with higher-order brain regions to regulate assignment matched based on body weight): Group 0 + (n = 16) or Group 24 + memory and cognition. These ﬁndings have direct clinical rele- (n = 11). After asymptotic discrimination was reached, animals were then sub- divided (matched based on body weight and performance over the last 8-trial block vance, as common bariatric surgeries partially denervate vagal 20, 53, 54 of training) into two additional groups to receive SDV (Group 0 + , n = 7; Group signaling and chronic vagus nerve blockade (VBLOC) was 24 + , n = 5) or sham (Group 0 + , n = 8; Group 24 + , n = 6) surgery 4 days after recently Food and Drug Administration-approved for obesity the last training day. After 7 days of post-surgery recovery, the animals were tested treatment . Future studies investigating the neuroendocrine and on deprivation discrimination performance for one 8-trial block (see Supplemen- tary Table 1). Seven days after deprivation discrimination testing, animals in neural pathways conveying energy-relevant signals between the Cohort 1 were tested in the zero maze task. Cohort 2 (SDV n = 8; sham n = 8) was GI tract and the HPC (and other regions of the telencephalon and tested in the spatial working memory task (5 days, described below) 7 days post cortex) will provide additional insight into the complex role of surgery. Five to 6 days later, dHPC tissue was harvested from Cohort 2 for gut-to-brain communication in cognitive control. immunblot analyses of BDNF and DCX (SDV, n = 8; sham, n = 8) (see Supple- mentary Methods). Cohort 3 (SDV n = 6, sham n = 9) was tested in the NOIC (5 days) task 14 days post surgery, followed by STFP 21 days post surgery (STFP; Methods 3 days). For STFP (described below), the SDV and sham groups were observers, Animals. Male Sprague–Dawley rats (Envigo; 320–450 g on arrival) were indivi- whereas demonstrator rats (non-operated, n = 8) were housed in a separate room dually housed with ad libitum access (except where noted) to water and chow from the observers. Based on results from our SDV experiments, CCK-SAP (n = 9) (LabDiet 5001, LabDiet, St. Louis, MO) on 12 h:12 h light/dark cycle (lights on at and SAP (control, n = 8)] rats were tested in the Barnes task (beginning 7 days post 08:00 h). All procedures involving animals were approved by the University of surgery; matching the timeline of post surgery SDV Barnes testing) followed by the Southern California Institute of Animal Care and Use Committee. NOIC task (beginning 14 days post surgery; matching the timeline of post surgery SDV NOIC testing). Seven days after NOIC testing, animals were tested in the zero maze task followed by the open ﬁeld task 3 days later. Five to 6 days later, dHPC Total SDV. Rats were habituated to liquid diet (Research Diets; AIN76A) for ﬁve tissue was harvested for immunblot analyses of BDNF (CCK-SAP, n = 8; SAP, n = days before surgery. Following a 24 h fast and under ketamine (90 mg/kg), xylazine 7) and DCX (CCK-SAP, n = 8; SAP, n = 8) (see Supplementary Methods; see (2.7 mg/kg), and acepromazine (0.64 mg/kg) anesthesia and analgesia (Metacam 2 Supplementary Fig. 3 for uncropped scan of CCK-SAP vs. SAP BDNF blot). mg/kg), the trunks of the subdiaphragmatic vagus nerve were transected as Groups were assigned matched according to body weight at the beginning of each described previously . A midline abdominal incision was made and then the experiment. For all video analyses for behavioral variables, experimenters were stomach was retracted caudally and the liver was retracted cranially to expose the blinded to group assignments of the animals. esophagus. The dorsal and ventral branches of the vagus were then dissected from the esophagus. Each vagal branch was ligated twice with a surgical thread at an interval of 1–2 cm, and then cauterized between the ligatures. In sham surgeries, Novel object in context. NOIC incorporates object recognition within a speciﬁc the trunks were exposed but the vagus nerve was not ligated or cauterized. The context, a test of contextual episodic memory. Procedures followed those pre- 15, 62 incision was then closed with running sutures along the abdominal wall and stop viously described . Rats undergo 2 days of habituation: half of the rats sutures along the skin. Rats were allowed to recover until liquid diet intake sta- (counterbalanced within surgical groups) are able to freely explore Context 1, a bilized (at least 1 week) and were then returned to a standard chow diet. After semi-transparent box (15 in W × 24 in L × 12 in H) with orange stripes, for 5 min, behavioral testing, SDV was veriﬁed functionally with i.p. CCK-induced food whereas the other half are habituated to Context 2, a gray opaque box (17 in W × 56, 57 intake reduction as described . Brieﬂy, the functional veriﬁcation consists of 17 in L × 16 in H). The following day, groups are switched and habituated to the analysis of food intake following i.p. CCK-8 (2 μg/kg; Bachem) or saline injections other context under the same conditions. Twenty-four hours later, on day 1 of 12 NATURE COMMUNICATIONS (2018) 9:2181 DOI: 10.1038/s41467-018-04639-1 www.nature.com/naturecommunications | | | NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04639-1 ARTICLE NOIC, each animal is exposed to two distinct objects: a 500 ml jar ﬁlled with blue powdered chow (ﬂavored with 2.5% marjoram or 0.5% thyme; counterbalanced) for water (object A) and a square glass container (object B) in Context 1. The next day, 30 min in a separate room from observers. Consistent with previous published work , the animals are placed in Context 2 with duplicates of object A. Twenty-four hours our pilot studies demonstrated that animals equally prefer these ﬂavors of chow. The later the animals are placed in the previous day’s location (Context 2) with objects demonstrator is then immediately placed in the social interaction arena with one of A & B and investigation of both objects is measured. Each day consists of 5 min their two assigned observer rats and allowed to socially interact for 30 min, followed sessions and contexts are cleaned with 10% ETOH between each animal. immediately by a 30 min interaction with the second assigned observer rat (observer Exploration is deﬁned as snifﬁng or touching the object with the nose or forepaws. rat order counterbalanced across experimental groups). Immediately after social The task scoring involves recording of the time spent exploring an object novel to interactions, observers are then returned to their home cages and allowed to eat ad the context vs. time spent exploring an object familiar to the context, and calcu- libitum for 1 h. Twenty-four hours later, the 23 h food-restricted observers are tested lation of a novelty or DI is based on these measurements and calculated as for STFP by placing two jars in the home cage: one containing paired ﬂavor (ﬂavored [Exploration of object B/(Exploration of object A + object B)]. Rats normally will chow that was given to the demonstrator) and the other with non-paired ﬂavor (novel preferentially investigate object B given that object B is a familiar object that is now ﬂavored chow not given to the demonstrator animal). Rats are then allowed to eat for presented in a novel context. NOIC is hippocampal dependent, as performance is 1 h and % preference for the paired ﬂavor is calculated by: 100*Demonstrator-paired impaired in rats with reversible muscimol-induced inactivation of the dorsal HPC ﬂavored chow intake/Demonstrator+ Novel ﬂavored chow intake. Cumulative food CA1 . intake for paired and non-paired ﬂavors are calculated. Untreated control animals learn to prefer the demonstrator-paired ﬂavor based on social interaction and olfactory food cues from the breath of the demonstrator rat; however, lesioning the dorsal HPC Spatial working memory. Spatial working memory is a component of short-term impairs consolidation of this preference learning. memory involving encoding and remembering information about one’s environ- ment and spatial orientation. The spatial working memory task used in the present Zero maze. Seven days following memory test procedures, all groups of rats (SDV, study involves an elevated white circular Barnes maze (Diameter: 122 cm, Height: sham, CCK-SAP, and SAP) were tested in the zero maze task to examine anxiety- 140 cm) with 18 holes evenly spaced around the outer edge of the table’s cir- like behavior. The zero maze is an elevated circular track, divided into four equal cumference. A hidden black escape box (38.73 cm × 11.43 cm × 7.62 cm) is placed length sections. Two sections were open with 3 cm high curbs, whereas the two under one of the holes. There are four sets of visuospatial cues (black and white other closed sections contained 17.5 cm high walls. Animals were placed in the stripes, a white circle, a red triangle, and an assortment of irregular shapes) placed maze for 5 min before being returned to the home cage. After the trial, the maze on each of the walls surrounding the table. One day after the habituation session, was cleaned with 10% ethanol. Innate anxiety was measured as the number of open spatial working memory training begins. Rats utilize visuospatial cues to learn the section entries and total time spent in open sections, deﬁned as the head and front location of the escape box, while being exposed to mildly aversive stimuli (120 W two paws in open sections. Previous studies have demonstrated that animals with bright overhead light, 75 db white noise), which motivates them to ﬁnd the escape high levels of innate anxiety (based on anxiolytic and anxiogenic experimental box to avoid these stimuli . Each rat receives two trials per day for ﬁve training manipulations) spend less time in the open sections compared with animals with days. The two trials are separated by 2 min (during which the maze is cleaned and low levels of innate anxiety . rotated). Importantly, the escape box is placed in the same location for both trials that occur on each individual training day, but is placed in a new location at the beginning of the ﬁrst trial for each subsequent training day. Errors are measured as Open ﬁeld. Three days following zero maze procedures, the CCK-SAP rats were investigation of holes that do not contain the escape box, and spatial working then tested in the open ﬁeld task, another behavioral paradigm used in rodents to memory is measured as the difference in the number of errors from trial 2 to trial 1 67 42 evaluate innate anxiety-like behavior . Open ﬁeld procedures are derived from . on each separate training day (which is then averaged over the ﬁve training days). The apparatus consists of a gray Plexiglas arena (60 cm × 56 cm), and a designated Control rats (Controls, Fig. 2b below) show improved performance (fewer errors) center zone within the arena (19 cm × 17.5 cm), placed under diffused lighting (30 st on the second trial of each training day compared to the 1 , indicating that the lux). Animals are placed in one of the bin’s four corners and allowed to freely spatial location of the escape hole can be integrated into working memory capacity explore for 10 minutes. The apparatus is cleaned with 10% ethanol between rats. in control rats. Innate anxiety was measured as the number of entries into the center zone, distance moved in center zone, and total distance moved in the entire arena. Previous work has shown that animals with innate anxiety-like behavior will spend less time in the Deprivation intensity discrimination. Deprivation intensity discrimination center zone and more time in close proximity to the walls compared with animals involves learning to use different levels of food restriction as interoceptive dis- with low levels of innate anxiety . criminative stimuli for sucrose reinforcement. The behavioral paradigm follows 6, 31, 64 that from previous publications . Training: Rats are divided into Group 0 + or Group 24 + and food deprivation levels alternate each day between 0 and 24 h c-Fos protein and mRNA expression. Nonsurgicated rats (n = 11) received i.p. for the entire experiment. Group 0 + receives a reinforcement of ﬁve sucrose injections of either saline (n = 5) or CCK (n = 6) (CCK-8, 8 μg/kg; Bachem), 90 pellets (45 mg, Product F06233, Bio-Serv) at the end of 4 min training sessions that min before perfusion and tissue was collected and processed as described above for take place under 0 h food deprivation, and receive no pellets during training ses- IHC and FISH processing. IHC detection of c-Fos was performed using rabbit anti- sions that take place under 24 h deprivation. Group 24 + receives the opposite c-Fos primary antibody (1:500, Cell Signaling, Catalog number: 2250 s) followed by contingency between food deprivation level and pellet delivery. During sessions in a donkey anti-rabbit IgG-AlexaFluor AF488 secondary antibody (1:500, Jackson which rats were trained under their non-rewarded deprivation condition, the Immunoresearch, RRID: AB_2340619). In the same animals, mRNA detection feeders operated but no pellets were delivered. The rats were then given 2 min to followed FISH procedures (see Supplementary Methods) and used c-Fos (ACD, consume the pellets before being removed from the conditioning chambers and Catalog number: 403591), VGLUT1 (ACD, Catalog number: 317001), or GAD2 returned back to their home cages. Training sessions always occurred at the same (ACD, Catalog number: 435801) probes. Representative images and quantiﬁcation time of day (10:00 h), but not every day to avoid a single-alternating schedule of for c-Fos protein and mRNA expression in dCA3 and DG obtained from both i.p. pellet delivery. The index of learning in the deprivation intensity discrimination saline and CCK injected animals were conﬁned to Swanson Atlas level 28–30 . task is food-anticipatory responding (head pokes in sucrose pellet delivery location; detected with photobeam interruptions/magazine entries) during the 1 minute Co-injection neural tracing. Neural pathway tracing experiments utilized two prior to pellet delivery. More speciﬁcally, the mean of the percentage of the three tracing techniques: co-injection monosynaptic neural tracing to identify second- 20 s intervals during the last minute of the session in which the magazine pho- order relay connections (COIN) and Cre-mediated dual-synaptic anterograde tobeam was interrupted was calculated as the dependent variable (as we’ve pre- 6 tracing (below) . Iontophoresis was performed using a precision current source viously described ). Rats with HPC lesions are impaired in learning and retention 6, 65 (Digital Midgard Precision Current Source, Stoelting) as described previously . of this discrimination problem , indicating that the HPC is critical for this type Rats (n = 14) received unilateral iontophoretic injections of AAV1-TurboRFP of learning process. Training consisted of six eight-trial blocks (eight trials for each (AAV1-hSyn-TurboRFP-WPRE-rBG; Penn Vector Core, Catalog number: deprivation state per training block), and testing (same procedures as training) V5574L) targeting the mNTS and, in the same animal, CTB-488 (CTB, AlexaFluor consisted of one eight-trial block 7 days after surgical recovery. 488 conjugate; ThermoFisher, Catalog number: C22841) targeting the dCA3. Following a 12-day survival period, animals were ﬁxation-perfused and tissue was Social transmission of food preference. STFP learning involves the utilization of collected and processed as described in Supplementary Methods. The native olfactory cues experienced during a social encounter to guide subsequent preference ﬂuorescent signal was ampliﬁed using a cocktail of mouse anti-CTB (1:500, Abcam, for a scented food. STFP task procedures were adapted from .Brieﬂy, demonstrators Catalog number: ab62429) and rabbit anti-RFP (1:2000, Rockland, RRID: (non-treated rats) and observers (surgicated rats, controls and experimental) are ﬁrst AB_2209751) primary antibodies followed by a cocktail of donkey anti-mouse IgG- habituated to an unscented powdered rodent chow [LabDiet 5001 (ground pellets)] AF488 secondary antibody (1:500, Jackson Immunoresearch, RRID: AB_2341099) overnight. Twenty-four hours later, observers are then assigned to demonstrators (1 and donkey anti-rabbit IgG-Cy3 (1:500, Jackson Immunoresearch, RRID: demonstrator assigned for 2 observers, counterbalanced by observer group) and are AB_2307443). Detection of AAV1 and CTB was by visualization of red (TurboRFP habituated to social interaction by being placed in a social interaction arena (23.5 cm and Cy3) and green (AlexaFluor 488) ﬂuorescence. In 3 of the 14 animals, injection W × 44.45 cm L × 27 cm H; clear plastic bin with Sani-chip bedding covering the sites were conﬁned to the mNTS (AAV1) and dCA3 (CTB) in the same animal. bottom) to interact with each other for 30 min. The next day following a 23 h period of Similar neuroanatomical labeling was observed in each of these three animals and food restriction for all rats, demonstrators are given access to one of two ﬂavors of representative images were obtained from one of these animals. Experimental NATURE COMMUNICATIONS (2018) 9:2181 DOI: 10.1038/s41467-018-04639-1 www.nature.com/naturecommunications 13 | | | ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04639-1 controls (n = 11) were included when neither anterograde nor retrograde labeling 3. Min, D. K., Tuor, U. I. & Chelikani, P. K. Gastric distention induced was observed in the MS following injection sites adjacent to (respectively) the functional magnetic resonance signal changes in the rodent brain. mNTS or dCA3 that did not include these regions. Neuroscience 179, 151–158 (2011). 4. Min, D. K., Tuor, U. I., Koopmans, H. S. & Chelikani, P. K. Changes in differential functional magnetic resonance signals in the rodent brain elicited Cre-mediated multisynaptic anterograde tracing. Rats (n = 16) received a by mixed-nutrient or protein-enriched meals. Gastroenterology 141, unilateral iontophoretic co-injection of AAV-Cre (4:5; AAV1-hSyn-Cre-WPRE- 1832–1841 (2011). hGH; Penn Vector Core, Catalog number: CS1087) and, to determine injection site, 5. Wang, G.-J. et al. Gastric stimulation in obese subjects activates the CTB-488 (1:5; ThermoFisher, Catalog number: C22841) in the mNTS. Next, the hippocampus and other regions involved in brain reward circuitry. Proc. 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NATURE COMMUNICATIONS (2018) 9:2181 DOI: 10.1038/s41467-018-04639-1 www.nature.com/naturecommunications 15 | | |
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