Hippocampus ghrelin signaling mediates appetite through lateral hypothalamic orexin pathways
Feeding behavior rarely occurs as a direct result of an immediate metabolic or nutrient deficiency . Rather , most animals ( including humans ) have habitual feeding patterns , consuming a fixed number of meals at approximately the same time each day . Environmental stimuli associated with learned meal anticipation elicit preparatory biological ‘cephalic’ responses ( e . g . , pancreatic insulin release ) that occur prior to food consumption ( Woods and Ramsay , 2000; Woods et al . , 1970 ) . These types of conditioned biological responses are advantageous when environmental factors place constraints on eating ( e . g . , predator threat , limited periods of food availability , and social or professional obligations ) because they promote consumption of a large quantity of food within a relatively short time period . One important biological signal associated with conditioned appetite and feeding is ghrelin , a hormone derived from P/D1 cells in the stomach that is the only known circulating hormone that increases food intake ( Tschöp et al . , 2000; Cowley et al . , 2003 ) . While often referred to as a ‘hunger’ hormone , ghrelin is perhaps more aptly described as a meal-anticipatory hormone based on findings from both humans and rodents showing that ghrelin levels rise before an anticipated feeding bout independent of energy deprivation levels ( Frecka and Mattes , 2008; Cummings et al . , 2001; Drazen et al . , 2006 ) . Genetic deletion of the ghrelin receptor ( growth hormone secretagogue receptor 1A; GHSR ) eliminates food anticipatory activity under meal entrainment feeding schedules ( Blum et al . , 2009; Davis et al . , 2011 ) , further supporting the notion that ghrelin acts as a conditioned meal-anticipatory signal . The mechanisms through which ghrelin communicates with the central nervous system to control learned aspects of food intake regulation are poorly understood . The hippocampus is a brain region historically associated with visuospatial and episodic memory function that has more recently been linked with the control of various feeding-relevant behaviors ( Benoit et al . , 2010; Davidson et al . , 2007; Kanoski , 2012; Parent et al . , 2014 ) , including conditioned meal anticipation ( Poulin and Timofeeva , 2008; Inglis et al . , 1994; Henderson et al . , 2013 ) . GHSR is expressed abundantly in hippocampal neurons ( Mani et al . , 2014 ) , and we recently demonstrated that pharmacological activation of GHSR by ghrelin in ventral/temporal subregions of the hippocampus ( vHP ) potently stimulates food intake by increasing both meal frequency and meal size ( Kanoski et al . , 2013 ) . However , the extent to which endogenous vHP GHSR signaling controls conditioned anticipatory feeding behavior has not been investigated . The lateral hypothalamic area ( LHA ) is implicated in the control of fundamental motivated behaviors , including food intake ( For review see: [Stanley et al . , 2011; Leinninger , 2011; Berthoud and Münzberg , 2011] ) . Classic studies show that LHA electrical ablation results in profound hypophagia ( Teitelbaum and Epstein , 1962; Anand and Brobeck , 1951 ) , whereas LHA electrical stimulation robustly increases food intake ( Delgado and Anand , 1953 ) . The LHA has direct connections with brain regions involved in the regulation of energy balance , including the hypothalamic arcuate and paraventricular nuclei ( Hahn and Swanson , 2015 ) , and the medial nucleus of the solitary tract ( Ciriello et al . , 2013 ) . In addition , the LHA is also directly connected with several brain regions that regulate ‘higher-order’ cognitive aspects of feeding behavior , including the vHP , amygdala , nucleus accumbens ( ACB ) , and ventral tegmental area ( VTA ) ( Hahn and Swanson , 2015; Hahn and Swanson , 2010; Cenquizca and Swanson , 2006; Hahn and Swanson , 2012; Goto et al . , 2005 ) . Of note , vHP pyramidal neurons in field CA1 and the subiculum are a major source of direct and exclusively ipsilateral input to the LHA , especially the dorsal perifornical LHA ( dpLHA ) ( Hahn and Swanson , 2015; Cenquizca and Swanson , 2006; Hahn and Swanson , 2012; Kishi et al . , 2000 ) . The dpLHA also has abundant expression of the neuropeptide orexin ( ORX/hypocretin ) ( Swanson et al . , 2005; Hahn , 2010 ) , which in addition to an established role in behavioral arousal ( Hagan et al . , 1999; Chemelli et al . , 1999 ) , also has a central orexigenic role in the control of ingestive behavior ( Willie et al . , 2001; Rodgers et al . , 2002; Sakurai , 2014; Thorpe et al . , 2005; Sweet et al . , 2004; Olszewski et al . , 2009 ) . We hypothesize that LHA ORX neurons are a critical downstream substrate of vHP ghrelinergic appetitive neural responding , because [1] orexin receptor 1 ( Ox1R ) signaling is required for ventricular ghrelin-induced hyperphagia ( Cone et al . , 2014 ) , and [2] LHA ORX neurons are activated in anticipation of feeding under meal entrainment schedules ( Jiménez et al . , 2013 ) . In the present study we investigate the neural substrates through which ghrelin communicates conditioned appetite and feeding behavior to and within the brain . We hypothesize the existence of a neural pathway whereby peripherally-derived ghrelin ( secreted in anticipation of feeding ) activates GHSR-expressing vHP glutamatergic ( pyramidal ) neurons , which in turn activate orexin-expressing dpLHA neurons to stimulate appetite and feeding via CNS Ox1R signaling . We employed a combination of methods to facilitate a correlative approach , including neural pathway-tracing , immunohistochemistry , behavioral neuropharmacology , meal entrainment conditioning , and excitotoxic unilateral lesion-mediated neural ‘disconnection’ between pharmacological vHP GHSR activation and the dpLHA . The results of these experiments suggest novel mechanisms linking mnemonic processes with gut-brain communication to control feeding behavior .
Figure 1 depicts a representative 100nl vHP injection site . Rats restricted to 4 hr chow access per day ( meal entrainment schedule ) learned to gradually increase their chow consumption over the 8 day training period ( significant main effect of Day , p<0 . 01 ) ( Figure 2A ) . Bilateral parenchymal delivery of the selective GHSR antagonist , JMV2959 ( 10 μg ) to vHP neurons significantly reduced 4 hr chow intake in meal-entrained rats ( p<0 . 05; Figure 2B ) . However , the same dose of JMV2959 had no effect on 4 hr rebound feeding following 20 hr food restriction in control rats that were not meal entrained ( Figure 2C ) . Moreover , the JMV2959-induced intake reduction observed in meal-entrained rats appears to be specific to conditioned feeding behavior as opposed to being based on higher baseline kcal consumption in meal entrained vs . control rats . This is supported by results showing that vHP JMV2959 administration did not reduce 4 hr Western diet intake in non-meal entrained , non-food restricted rats under conditions in which total kcal intake exceeded levels observed in meal-entrained rats ( Figure 2D ) . Collectively these findings indicate that endogenous vHP ghrelin signaling is critical in the control of conditioned feeding behavior . 10 . 7554/eLife . 11190 . 003Figure 1 . Representative vHP injection site is shown as localization of pontamine sky blue ink following a 100 nl injection . CA1sp , CA3sp: CA1 and CA3 pyramidal cells; alv = alveus . DOI: http://dx . doi . org/10 . 7554/eLife . 11190 . 00310 . 7554/eLife . 11190 . 004Figure 2 . Effects of vHP GHSR blockade on conditioned food intake . ( A ) Daily 4 hr chow intake during meal entrainment acquisition , where animals were limited to 4 hr food intake/day for 8 days . ( B ) 4 hr chow intake in meal-entrained animals following bilateral vHP administration of JMV2959 . ( C ) 4 hr chow intake in non-meal entrained , 20 hr food-restricted rats following bilateral vHP administration of JMV2959 . ( D ) 4 hr Western diet intake in non-meal entrained , non-food deprived rats following bilateral vHP administration of JMV2959 . Data are mean ± SEM; *p<0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 11190 . 00410 . 7554/eLife . 11190 . 005Figure 2—figure supplement 1 . Effects of LHA GHSR blockade on conditioned food intake . ( A ) Daily 4 hr chow intake during meal entrainment acquisition . ( B ) 4 hr chow intake in meal-entrained animals following bilateral LHA administration of JMV2959 . ( C ) 4 hr chow intake in non-meal entrained , 20 hr food-restricted rats following bilateral LHA administration of JMV2959 . Data are mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 11190 . 005 Like the vHP , the LHA is another brain region that expresses GHSR , and activation of these receptors increases feeding ( Mani et al . , 2014; Olszewski et al . , 2003 ) , Moreover , orexin-producing neurons in the LHA are activated by external food-related cues and by LHA administration of ghrelin ( Olszewski et al . , 2003; Petrovich et al . , 2012 ) . Thus , to determine whether endogenous LHA ghrelin signaling also contributes to conditioned feeding responses , we repeated the meal entrainment and non-entrained , 20 hr food deprivation procedures in LHA cannulated rats . Animals restricted to 4 hr chow access per day again learned to increase their chow consumption over the 8 day training period ( significant main effect of Day , p<0 . 01 ) ( Figure 2—figure supplement 1A ) . Bilateral delivery of JMV2959 had no effect on 4 hr chow intake in meal entrained rats , nor did LHA JMV2959 application influence 4 hr chow intake in 20 hr food deprived , non-meal entrained rats ( Figure 2—figure supplement 1B , C ) . These findings suggest that unlike vHP ghrelin signaling , LHA ghrelin signaling does appear to be critical for the manifestation of conditioned feeding behavior . A vHP to LHA pathway hypothesized to play a role in GHSR signaling for the control of conditioned feeding behavior , was investigated using retrograde pathway-tracing and immunohistochemistry . Following injection of either Fluorogold or cholera toxin B subunit ( CTB ) into the dpLHA , abundant exclusively ipsilateral retrograde labeling was seen in the vHP field CA1 and subiculum ( i . e . , no Fluorogold- or CTB-immunoreactive cells were observed in the ventral CA1 contralateral to tracer injections ) , consistent with previous findings ( Hahn and Swanson , 2010; Hahn and Swanson , 2012; Cenquizca and Swanson , 2006 ) . Further investigation of CTB and Fluorogold retrogradely-labeled vHP neurons found that most were also immunoreactive for GHSR [representative dpLHA CTB injection site depicted in Figure 3A; retrogradely-labeled CTB immunoreactive soma in the vHP depicted in Figure 3C; inset location delineated in Figure 3B] . Consistent with previous literature revealing extensive GHSR mRNA expression in the vHP CA fields ( Mani et al . , 2014; Zigman et al . , 2006 ) , our immunohistochemistry analyses confirmed robust GHSR protein expression in vHP CA1 pyramidal neurons ( Figure 3D ) . Quantification of the percentage of back-labeled CTB and Fluorogold immunoreactive soma also expressing GHSR revealed 84 . 11% ( SEM = 0 . 07 ) colocalization in rats injected with CTB in the LHA , and 86 . 22% ( SEM = 0 . 03 ) colocalization in rats that were injected with Fluorogold in the LHA ( representative CTB and GHSR co-labeled soma depicted in Figure 2E ) . These data indicate that the vast majority of vHP neurons that provide an ipsilateral input to the dpLHA also express GHSR . 10 . 7554/eLife . 11190 . 006Figure 3 . GHSR-expressing vHP CA1 neurons provide input to dpLHA neurons . ( A ) A representative dorsal perifornical LHA injection site of CTB-AF594 ( red ) . fx:fornix; V3h: Third ventricle ( B ) Darkfield cytoarchitecture of the vHP , where insets C-E represents field CA1 pyramidal layer ( CA1sp ) insets from the square ( white dashed line ) . ( C ) vHP retrogradely-labeled CTB immunoreactive perikarya ( red ) . ( D ) GHSR protein expression ( green ) . ( E ) CTB and GHSR co-labeled soma . DOI: http://dx . doi . org/10 . 7554/eLife . 11190 . 006 To explore the functional relevance of the exclusively unilateral vHP to dpLHA communication in vHP ghrelin-mediated hyperphagia , rats with unilateral excitotoxic ( NMDA ) or sham lesions in the dpLHA were implanted with bilateral vHP-targeted cannulae ( representative unilateral dpLHA lesion depicted in Figure 4 with NeuN immunohistochemistry ) . Food intake was measured after unilateral vHP administration of ghrelin ( 300 pmol ) to rats with dpLHA lesions or sham lesions that were either ipsilateral or contralateral to the side of ghrelin administration ( i . e . vHP to LHA communication either eliminated or intact ) . Ghrelin delivered to the vHP contralateral to the LHA lesion ( or sham lesion ) significantly increased chow intake in both groups , whereas ghrelin delivered to the vHP ipsilateral to the LHA lesion ( or sham lesion ) significantly increased food intake only in the Sham group ( Figure 5A–D; *indicates p<0 . 05 vs . vehicle treatment , Ŧ indicates p<0 . 07 vs . vehicle treatment ) . A repeated-measures overall ANOVA was also performed with ‘lesion group’ as a between subjects variable and ‘drug’ as a repeated measures variable . There was no overall significant ‘group’ effect on food intake at any point , whereas the overall ‘drug’ effect was significant at all time points ( p<0 . 001 ) . Furthermore , there was a significant interaction between ‘group’ and ‘drug’ at the 5 hr time point [F ( 1 , 15 ) =4 . 93 , p<0 . 05] , whereas this interaction just missed significance at the 1 hr and 3 hr time points . These results show that vHP neural communication with the dpLHA is required for vHP ghrelin-mediated feeding effects . 10 . 7554/eLife . 11190 . 007Figure 4 . Representative unilateral dpLHA lesions . ( A ) NeuN immunohistochemistry contralateral to a NMDA dorsal perifornical ( dp ) LHA lesion ( intact side ) . ( B ) NeuN immunohistochemistry for a representative unilateral NMDA dpLHA lesion targeting the dorsal perifornical LHA . DOI: http://dx . doi . org/10 . 7554/eLife . 11190 . 00710 . 7554/eLife . 11190 . 008Figure 5 . Effects of vHP-dpLHA unilateral disconnection on vHP ghrelin-mediated hyperphagia . Cumulative chow intake following unilateral vHP injections of ghrelin ( 300 pmol ) that was: ( A ) Ipsilateral to a unilateral dpLHA lesion , ( B ) ipsilateral to a dpLHA sham lesion . ( C ) contralateral to a dpLHA lesion , and ( D ) contralateral to a dpLHA sham lesion . Data are mean ± SEM; *p<0 . 05; Ŧ p<0 . 07 . DOI: http://dx . doi . org/10 . 7554/eLife . 11190 . 008 Anterograde pathway-tracing with Phaseolus vulgaris leucoagglutinin ( PHAL ) provided further support for a direct and exclusively ipsilateral connection from the vHP to the dpLHA ( Figure 6—figure supplement 1 displays PHAL immunoreactive axons that are exclusively ipsilateral to a vHP PHAL iontophoresis injection ) . PHAL was injected by iontophoresis into the vHP field CA1 ( representative injection site depicted in Figure 6A ) . Double-label immunohistochemistry for PHAL and ORX revealed several instances of PHAL-labeled axons with numerous varicosities in close apposition to ORX-expressing neurons within the dpLHA ( representative examples shown in Figure 6C , inset location delineated in Figure 6B ) . Subsequent triple-label immunohistochemistry for PHAL , ORX , and the presynaptic marker synaptophysin indicated that the apparent terminal appositions were putative sites of synaptic input from vHP CA1 neurons to ORX-expressing neurons ( representative examples shown in Figure 6D ) . These data confirm an input to the dpLHA from the vHP field CA1 , and indicate further that this input targets ORX-expressing neurons . 10 . 7554/eLife . 11190 . 009Figure 6 . vHP CA1 neurons provide input to dpLHA ORX-expressing neurons . ( A ) A representative PHAL ( blue ) injection site centered in ventral hippocampus field CA1 . CA1sp , CA3sp: CA1 and CA3 pyramidal cells . ( B ) Darkfield microscopy , where neuroanatomical analyses were performed in the dpLHA ( fx: fornix; V3h: Third ventricle; location shown in C ) . Inset ( C ) confocal imaging reveals some orexin neurons ( red ) in the dpLHA that are in close apposition to vHP field CA1-originating axons , labeled with PHAL ( blue ) . ( D ) Triple-label imaging reveals co-labeling of PHAL and synaptophysin ( green ) in axons in apposition to dpLHA orexin-expressing soma ( indicated by white arrows ) . DOI: http://dx . doi . org/10 . 7554/eLife . 11190 . 00910 . 7554/eLife . 11190 . 010Figure 6—figure supplement 1 . CA1 vHP neurons project ipsilaterally to the dpLHA . ( A–C ) PHAL-immunoreactive axons originating from the vHP ( field CA1 ) within the dpLHA , revealing an exclusively ipsilateral projection from the vHP to the dpLHA ( v3h = 3rd ventricle; fx = fornix ) . DOI: http://dx . doi . org/10 . 7554/eLife . 11190 . 010 Unilateral vHP ghrelin ( 300 pmol ) administration robustly increased expression of the neural activation marker Fos in the ipsilateral LHA compared to vehicle treatment ( p<0 . 01 vs . vehicle and vs . contralateral LHA ) . Notably , vHP ghrelin injection activated approximately 70% of orexin-expressing neurons in the ipsilateral LHA ( Figure 7; p<0 . 01 compared to vehicle treatment ) . These data are consistent with the hypothesis that vHP ghrelin signaling activates orexin-expressing dpLHA neurons . 10 . 7554/eLife . 11190 . 011Figure 7 . vHP GHSR activation increases Fos expression in dpLHA orexin-expressing neurons . ( A ) Orexin-expressing neurons ( red ) in the dpLHA; fx: fornix . ( B ) Fos expression ( green ) in the dpLHA induced by ipsilateral vHP ghrelin ( 300 pmol ) administration . ( C ) Co-labeling of dpLHA orexin expression and Fos expression . ( D ) Quantification of orexin positive soma co-labeled with Fos , where co-labeling was significantly higher compared to vHP vehicle injections . Data are mean ± SEM; *p<0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 11190 . 011 To test whether vHP ghrelin-mediated hyperphagia requires downstream Ox1R signaling , rats first received a lateral ventricle ( LV ) injection of either vehicle ( DMSO ) or the Ox1R antagonist , SB334867 , at a dose ( 30 nmol ) that was subthreshold for intake effects alone under these conditions . Immediately afterwards , the rats received unilateral parenchymal vHP injections of ghrelin ( 300 pmol ) or vehicle on separate test days . Treatments were counter-balanced using a within-subjects design . Ghrelin significantly increased chow intake in rats pretreated with vehicle in the LV , but had no effect relative to vehicle on intake in rats pretreated with SB334867 ( Figure 8A , B; *indicates p<0 . 05 vs . vehicle treatment , Ŧ indicates p<0 . 07 vs . vehicle treatment ) . These data indicate that vHP-mediated feeding effects involve downstream Ox1R signaling . 10 . 7554/eLife . 11190 . 012Figure 8 . Effects of Orexin-1 receptor blockade on ghrelin-mediated hyperphagia . Cumulative chow intake following: ( A ) pre-treatment with lateral ventricle ( LV ) vehicle followed by vHP ghrelin ( 300 pmol , unilateral ) administration or ( B ) LV Ox1R antagonist , SB334867 ( 30 nmol ) , followed by vHP ghrelin administration . Data are mean ± SEM; *p<0 . 05; Ŧ p<0 . 07 . DOI: http://dx . doi . org/10 . 7554/eLife . 11190 . 012
Ghrelin levels rise in anticipation of feeding ( Frecka and Mattes , 2008; Cummings et al . , 2001; Drazen et al . , 2006 ) and ghrelin receptor null mice show blunted meal anticipatory responding under meal entrainment feeding schedules ( Blum et al . , 2009; Davis et al . , 2011 ) . Compared to equivalent levels of food restriction , the anticipation of eating in meal entrained rats had a greater stimulatory effect on ghrelin release ( Drazen et al . , 2006 ) , indicating that ghrelin acts as a meal anticipation hormonal signal that facilitates the consumption of large quantities of food . This notion is also consistent with data showing a role for ghrelin in stimulating gastric motility ( Masuda et al . , 2000; Peeters , 2003 ) and increasing meal size ( Kanoski et al . , 2013; Lee et al . , 2011; Swartz et al . , 2014; de Lartigue et al . , 2010; Faulconbridge et al . , 2003 ) . Here we identify and characterize a novel neural substrate extending these previous findings by demonstrating that vHP ghrelin signaling is physiologically relevant for conditioned feeding behavior . After exposure to a meal entrainment schedule in which rats were restricted to 4 hr food access each day , pharmacological blockade of vHP GHSR signaling prior to food access significantly reduced 4 hr chow consumption . Comparatively , vHP GHSR blockade had no effect on a 4 hr chow intake in equally food-restricted rats that had not undergone the meal entrainment learning . Moreover , the intake reduction appears to be specific to conditioned feeding behavior as opposed to being based on higher baseline kcal consumption in meal entrained vs . control rats . This is supported by results showing that vHP JMV2959 administration did not reduce Western diet intake in non-food deprived , non-meal entrained animals under conditions where baseline kcal intake of the Western diet was actually higher than in meal-entrained rats consuming chow . However , another study showed that peripheral GHSR antagonist ( Compound 26 ) delivery failed to suppress feeding in meal-entrained mice ( Perello et al . , 2010 ) , yet we note that this previous study utilized a different GHSR antagonist , a different species , and a different route of administration ( peripheral ) . Collectively , these findings build on previous work showing that pharmacological vHP GHSR activation increases meal frequency and size ( Kanoski et al . , 2013 ) . Here we establish the endogenous relevance of vHP GHSR signaling to a specific component of feeding – conditioned feeding . We hypothesize that pharmacologically induced hyperphagia following vHP ghrelin injections occurs , at least in part , by augmenting the saliency of conditioned food-relevant interoceptive stimuli and/or external cues that are present in the environment . To investigate neural pathways downstream to vHP ghrelin-mediated feeding behavior , we focused on the dpLHA region that contains abundant ORX-expressing neurons ( Swanson et al . , 2005; Hahn , 2010 ) , and provide evidence of an input to dpLHA ORX-expressing from GHSR-expressing neurons in the vHP ( field CA1 ) . Our data confirm the existence of an exclusively ipsilateral direct input to the dpLHA from the vHP field CA1 , and further demonstrate that ~85% of vHP field CA1 neurons retrogradely labeled from the dpLHA are immunoreactive for GHSR . These findings suggest that vCA1 ghrelin-responsive neurons send a direct input to the dpLHA , thereby highlighting the dpLHA as a downstream target for vHP ghrelin-mediated feeding . Based on research showing an extensive population of orexin producing neurons in the dpLHA ( Swanson et al . , 2005; Hahn , 2010; Sakurai et al . , 1998; Chen et al . , 1999 ) , as well as functional and neuroanatomical interactions between ghrelin and Ox1R signaling ( Cone et al . , 2014; Olszewski et al . , 2003; Perello et al . , 2010; Solomon et al . , 2007; Toshinai et al . , 2003 ) , we next investigated the relationship between vCA1 and dpLHA orexin neurons . A direct connection from the vHP to dpLHA neurons was confirmed by the presence of PHAL-immunoreactive axons in the dpLHA following vHP field CA1 PHAL injections . Several instances of PHAL-labeled axons with varicosities and terminal boutons were recorded in close apposition to dpLHA ORX-expressing neurons . Triple-labeling for PHAL , ORX and the presynaptic marker synaptophysin indicated that some of these close appositions were putative sites of synaptic input . Next , we tested whether vHP GHSR activation engages dpLHA orexin neurons . Immunohistochemical analysis of Fos and orexin expression in the dpLHA in rats treated with vHP ghrelin or vehicle injections revealed Fos expression in approximately 70% of dpLHA orexin-expressing neurons . Taken together , these findings indicate a functional pathway involving GHSR-expressing vHP field CA1 neurons and their downstream communication to dpLHA orexin neurons . To investigate the functional relevance of a putative vHP field CA1 ghrelin to LHA orexin connection further , we combined behavioral neuropharmacology ( vHP ghrelin injection ) with an excitotoxic unilateral LHA lesion-mediated neural disconnection approach ( adapted after [Petrovich et al . , 2002] ) . This approach takes advantage of an exclusively ipsilateral connection between the vHP and the LHA . Results show that vHP ghrelin administration ipsilateral to unilateral LHA lesions ( vHP GHSR to LHA communication eliminated ) blocks vHP ghrelin-activated hyperphagia that occurs in rats with sham lesions , and that also occurs when vHP ghrelin injections are given contralateral to the unilateral dpLHA lesion ( vHP GHSR to LHA communication intact ) . These data indicate that vHP to dpLHA communication is critical for vHP ghrelin-mediated feeding effects . A limitation in interpreting these findings with regard to the control of conditioned feeding behavior is that these experiments utilized pharmacological activation of vHP ghrelin receptors during free feeding conditions , and therefore the endogenous relevance of this neural circuitry during conditioned or entrained feeding requires further research . However , our data from Exp . 1 provide strong support for the endogenous relevance of vHP ghrelin signaling system for conditioned food intake . We hypothesize that the robust hyperphagic response following vHP GHSR activation during free feeding conditions is based , at least in part , on pharmacologically activating conditioned feeding behavior in the presence of food-associated cues . In other words , pharmacological vHP GHSR activation is potentially bolstering the saliency of internal ( e . g . , circadian , metabolic ) or external cues within the animal’s environment ( e . g . , food hopper ) that have the capacity to promote feeding behavior based on learned associations with feeding . Consistent with this framework , vHP GHSR activation increases meal frequency in response to external environmental stimuli ( auditory , visual ) that were associated with palatable food access , whereas vHP ghrelin signaling does not augment feeding in response to control stimuli with no prior food association ( Kanoski et al . , 2013 ) . However , because present data do not directly assess the functional importance of vHP ghrelin to LHA orexin neural circuitry in conditioned feeding , we acknowledge that this pathway might also mediate aspects of non-conditioned appetite and feeding behavior . Indeed , ghrelin signaling in other brains regions appears to stimulate non-conditioned appetitive responding ( Skibicka et al . , 2011; Skibicka et al . , 2013 ) . For example , ICV or intra-amydala administration of the same GHSR antagonist used in this study ( JMV2959 ) reduces free feeding in fasted ( non-entrained ) animals ( Salomé et al . , 2009; Alvarez-Crespo et al . , 2012 ) . Neurons in the LHA also express GHSR , and LHA orexin-producing neurons are activated by external food-related cues as well as by direct LHA ghrelin administration ( Olszewski et al . , 2003; Petrovich et al . , 2012 ) . These findings suggest that in addition to the vHP , the LHA might also be a target for ghrelinergic conditioned feeding responses . However , our present data reveal that intra-LHA JMV2959 ( including a dose that was effective in the vHP ) had no effect on food intake in meal-entrained and non-meal-entrained animals , suggesting that while ghrelin signaling in various brain regions stimulates non-conditioned feeding behavior , vHP ghrelin signaling is a potentially unique neural substrate for learned aspects of feeding behavior . We note that it is also possible that vHP ghrelin to LHA orexin neural circuitry mediates non-conditioned aspects of feeding , however , this notion requires further investigation . Our neuroanatomical data are consistent with previous research showing that vHP output to the LHA primarily targets the dpLHA region ( Hahn and Swanson , 2010; Hahn and Swanson , 2012; Kishi et al . , 2000 ) that was targeted with our unilateral NMDA lesions . While the anatomical and behavioral data in the current study suggest that vHP neurons communicate ghrelinergic signaling to the dpLHA directly , we recognize that multisynaptic pathways are also likely be involved . In addition to providing an input to the LHA , vHP field CA1 neurons also provide an input to several other feeding-related brain regions , including the amygdala and medial prefrontal cortex ( mPFC ) ( Ishikawa and Nakamura , 2006; Cenquizca and Swanson , 2007 ) . These regions also provide a topographically organized input to the LHA ( Hahn and Swanson , 2015; Hahn and Swanson , 2012 ) , and neural connectivity between the amygdala-LHA and mPFC-LHA has been shown to be critical for conditioned cue-potentiated feeding ( Petrovich et al . , 2002; Petrovich et al . , 2005 ) . Additional studies are required to determine the functional relevance of these other pathways to vHP ghrelin-mediated appetite . We examined the requirement of downstream Ox1R signaling for vHP ghrelin-mediated feeding effects , demonstrating that central Ox1R blockade ( via LV injections of an Ox1R antagonist ) attenuated the hyperphagic effects of vHP ghrelin signaling . However , because ventricular injections spread throughout the CNS and Ox1R s are expressed throughout the brain , ( Hervieu et al . , 2001; Marcus et al . , 2001 ) , we can only speculate as to the mediating sites of Ox1R activation . Recent research has shown that blockade of Ox1Rs in the VTA attenuates food intake induced by LV ghrelin ( Cone et al . , 2014 ) . Given these findings , and the role of VTA dopaminergic signaling in various aspects of feeding behavior ( Cone et al . , 2014; Skibicka et al . , 2013; Nieh et al . , 2015; Liu and Borgland , 2015; Narayanan et al . , 2010; Cacciapaglia et al . , 2011 ) , we hypothesize that VTA Ox1R signaling is a critical downstream target of vHP ghrelin to dpLHA orexin signaling , however , this remains to be tested . Previous literature has also shown that central orexin signaling has roles in sleep/wake and arousal in addition to regulating feeding behavior ( Hagan et al . , 1999; Chemelli et al . , 1999 ) . The current study did not directly assess the relationship between vHP ghrelin signaling and orexin mediated arousal . It is possible that the enhanced saliency of external food-related cues following vHP ghrelin signaling is mediated by increased arousal , as well as potential changes to circadian activity , from downstream activation of central Ox1R's ( possibly in the locus coeruleus ) . Further studies are required to test these possibilities , as well as the role of downstream Ox1R signaling in other aspects of feeding behavior ( Zhu et al . , 2002; Goforth et al . , 2014; Zheng et al . , 2007; Parise et al . , 2011; Harris et al . , 2005; Cason and Aston-Jones , 2013; Thorpe and Kotz , 2005; Thorpe et al . , 2006 ) . Based on the present results and the findings of related previous research , we propose the following model in which vHP ghrelin signaling engages conditioned feeding behavior ( Figure 9 ) : [1] Ghrelin is secreted from the stomach in response to cues ( internal or external ) related to meal anticipation , [2] Ghrelin crosses the blood brain barrier and enters the central nervous system , [3] vHP GHSR-expressing neurons are activated by ghrelin , [4] These vHP ghrelin-activated neurons engage downstream dpLHA orexin-expressing neurons , [5] Activation of this neural pathway increases appetite and feeding behavior . Together , our data highlight novel neurobiological mechanisms by which ghrelin engages higher-order brain circuitry , that in turn controls hypothalamic systems necessary for the regulation of appetite . 10 . 7554/eLife . 11190 . 013Figure 9 . A model for vHP ghrelin-mediated conditioned feeding behavior . ( A ) Animals learn to anticipate a meal based on external or internal food cues . ( B , C ) Ghrelin secreted from the stomach in anticipation of feeding crosses the blood brain barrier and enters the central nervous system . ( D ) Ghrelin acts on GHSR in vHP neurons , which engages ( E ) Downstream activation of dpLHA orexin neurons and CNS Ox1R signaling . ( F ) Activation of this neural pathway increases conditioned appetite and feeding behavior . DOI: http://dx . doi . org/10 . 7554/eLife . 11190 . 013
Male Sprague-Dawley rats ( Harlan , Indianapolis , IN ) ( 320–450 g ) were individually caged in a temperature-controlled vivarium with unlimited access ( except where noted ) to water and food ( LabDiet 5001 , LabDiet , St . Louis , MO ) on either 12 hr:12 hr light/dark cycle ( lights on at 8:00 AM ) or 12:12 hr reverse light/dark cycle ( lights off at 10:00AM ) . For all surgical procedures , rats were anesthetized ( ketamine 90mg/kg and xylazine , 2 . 8 mg/kg ) , and sedated ( acepromazine . 0 . 72 mg/kg ) . All procedures were approved by the Institute of Animal Care and Use Committee , at the University of Southern California . Experiments involving central drug injections were as follows: ( Exp . 1 ) bilateral vHP injection of a ghrelin receptor ( GHSR ) antagonist ( JMV2959 , EMD Millipore , Billerica , MA ) ; ( Exp . 3 ) unilateral dpLHA injections of NMDA ( Sigma-Aldrich , St . Louis , MO ) ; ( Exp . 3 and 5 ) unilateral vHP injection of ghrelin ( Bachem , Torrance , CA ) ; ( Exp . 6 ) bilateral vHP injection of ghrelin and lateral ventricle injection of an orexin receptor type 1 ( Ox1R ) antagonist ( SB334867 , Tocris Bioscience , Ellisville , MO ) . Drugs were dissolved in artificial cerebrospinal fluid ( aCSF ) , except SB334867 which was dissolved in DMSO . Drugs were delivered using a microinfusion pump ( Harvard Apparatus , Holliston , MA ) connected to a 33-gauge microsyringe injector , via indwelling guide cannulae . Flow rate was calibrated and set to 5 µl/min; injection volume was 100 nl/hemisphere for Exp . 1 , 3 and 6 . Injection volume for NMDA lesions were 200 nl ( dose: 4 μg ) . Ventricular injections in Exp . 6 were 1 ul in volume . Injectors were left in place for 30 sec post-injection . Guide cannulae ( 26-gauge , Plastics One , Roanoke , VA ) for bilateral vHP drug injections were implanted at the following stereotaxic coordinates ( Paxinos and Watson , 2007 ) : -4 . 9 mm anterior/posterior ( AP ) , 4 . 8 mm medial/lateral ( ML ) , 6 . 1 mm dorsal/ventral ( DV ) ; they were then affixed to the skull with jewelers screws . Coordinates for lateral ventricular injections of Ox1R antagonist SB334867 were -0 . 9 mm AP , 1 . 8 mm ML , 2 . 6 mm DV . Coordinates for excitotoxic ( 4 ug , NMDA , 200 nl ) LHA lesions , were -2 . 9 mm AP , 1 . 1 mm ML , -8 . 9 mm DV . Injectors for drug administration projected 2 mm beyond guide cannulae . Coordinates for LHA injections were -2 . 9 mm AP , +/- 1 . 0 mm ML , -6 . 7 mm DV . Placement of vHP and LHA cannulae placement was determined post-mortem by injection ( via guide cannulae ) of a blue dye ( 100 nl 2% pontamine sky blue ink ) . Data from animals with dye restricted to the vHP were included in the analysis ( a representative vHP injection site is shown in Figure 1 ) . In total , 12% of rats with vHP cannulae were excluded from the analysis because of misplaced cannulae , determined by the presence of blue dye as noted above . The extent of unilateral NMDA dpLHA-targeted lesions was determined postmortem by immunohistochemical detection of the neuronal marker NeuN . Rats showing pronounced ( 65% ) loss of NeuN labeling within the dpLHA compared to a contralateral sham ( aCSF ) injection in the same animal were included in data analysis ( two rats with minimal or misplaced lesions were excluded ) . Targeting of the OX1R antagonist SB334867 to the lateral ventricle ( Exp . 6 ) was verified 1 week post-injection of the antagonist by elevation of cytoglucopenia resulting from injection ( at the same coordinates ) of 210 μg ( 2 μl ) of 5-thio-D-glucose ( Ritter et al . , 1981 ) . A post-injection elevation of at least 100% of baseline glycemia was required for subject inclusion ( 1 rat was excluded ) . Experiments involving neural pathway-tracing were as follows: ( Exp . 2 ) Retrograde tracing from the dpLHA following unilateral pressure injection of either Fluorogold ( FG , Fluorochrome LLC , Denver , CO; 2% in 0 . 9% NaCl ) , or cholera toxin B subunit conjugated to ( red fluorophore ) Alexa Fluor 594 ( CTB-AF594 , 0 . 25% in 0 . 9% NaCl , Life Technologies , Carlsbad , CA; Cat . No . C-22842 ) . Pressure injections were performed using a microinfusion pump as described above; injection volumes were 100 nl ( Exp . 4 ) . Anterograde tracing from the vHP field CA1 following unilateral iontophoretic injection of Phaseolus vulgaris-leucoagglutinin ( PHAL , Vector Labs , Burlingame , CA; 2 . 5% in 0 . 1 M sodium phosphate-buffered saline , pH 7 . 4 ) was performed using a precision current source ( digital midgard precision current source , Stoelting ) as described previously ( Hahn and Swanson , 2010 ) . PHAL immunoreactive axons in the LHA confirmed the exclusively ipsilateral nature of vHP to LHA projections ( Figure 6—figure supplement 1; representative vHP PHAL injection site , Figure 6 ) . Experiments 2–5 included immunohistochemical ( IHC ) analyses . Animals were anesthetized , then perfused transcardially with ice-cooled 0 . 9% saline , followed by 4% paraformaldehyde in 0 . 1 M borate buffer of pH 9 . 5 . The brains were removed and immersed in the same ( unused ) fixative containing 12% sucrose for 20–24 hr at 4ºC . The brains were then blocked transversely at the level of the caudal midbrain , and each block was flash-frozen in dry-ice cooled hexane before being sectioned frozen on a sliding microtome ( transverse plane , 30 µm thickness , 5 series ) . Sections were stored in antifreeze solution at -20°C until further processing ( Antifreeze solution: 30% ethylene glycol , 20% glycerol in 0 . 02 M potassium phosphate-buffered saline—KPBS ) . The following primary antibodies were used: ( Exp . 2 ) anti-CTB ( raised in goat ) and anti-GHSR ( goat ) ; ( Exp . 4 ) anti-PHAL ( goat ) , anti-ORX1a ( rabbit ) , and anti-synaptophysin ( mouse ) ; ( Exp . 5 ) anti-cFos ( sheep ) and anti-ORX1a ( rabbit ) . Antibody incubations were done at 4ºC ( washes and other steps at room temperature ) . Primary antibody incubation was overnight ( ~18 hrs ) . Primary antibodies were diluted in the following solution: KPBS containing 1% donkey serum and 0 . 1% Triton X-100; this step was followed by KPBS washes ( 8 changes , 10 mins each ) . Overnight secondary antibody incubation: Biotinylated secondary antibodies or secondary-fluorophore conjugates were diluted in KPBS containing 0 . 1% Triton X-100; this step was followed by KPBS washes ( 6 changes , 10 mins each ) . Overnight tertiary incubation ( if biotinylated secondary antibodies were applied: Streptavdin-fluorophore conjugate was diluted in KPBS; this step was followed by KPBS washes ( 3 changes , 10 mins each ) . Sections were mounted onto glass slides , allowed to dry , and coverslipped , using 50% glycerol in KPBS mountant . Coverslip edges were sealed with clear nail polish . Controls for IHC: For each primary antibody used , a standard control was performed in which the primary antibody was omitted from the protocol ( to control for non-specific signal resulting from subsequent antibody and signal-detection reagents ) . In addition , for GHSR primary antibodies , an adsorption / neutralization was performed using the GHSR peptide sequence ( a propriety sequence derived from a portion of the complete GHSR sequence ) . Following this adsorption , GHSR labeling was abolished . Photomicrographs were acquired using either a Nikon 80i and ( Nikon DS-QI1 , 1280X1024 resolution , 1 . 45 megapixel ) under epifluorescent illumination , or as optical slices using a Zeiss LSM 700 UGRB Confocal System ( controlled by Zeiss Zen software ) . All photomicrographs in the figures are oriented such that: up = dorsal , down = ventral , left = medial , right = lateral . Rats were implanted bilaterally with vHP-targeted cannulae ( n=11 ) . After recovery from surgery , they were put on a meal-entrainment schedule where they were limited to food access for 4 hr daily , beginning at 10:00 AM ( start of dark cycle ) , for 8 days . One day after the last training day , immediately prior to lights off , the meal-entrained rats were injected via their bilaterally implanted cannulae with either 0 µg , 1 µg , or 5 µg of a ghrelin receptor ( GHSR ) antagonist ( JMV2959; total doses: 0 µg , 2 µg , 10 µg ) . Dose selection for JMV2959 was based on previous reports ( Skibicka et al . , 2011; Alvarez-Crespo et al . , 2012 ) . Treatments were separated by 2–3 intervening day using a counterbalanced within-subjects design . Food intake was recorded 4 hr post-injection ( food spillage accounted for ) . To control for the effects of vHP GHSR antagonism on food intake independent of meal-entrainment , 2 additional groups of rats ( n=8/group ) were 20 hr food deprived before receiving vHP injections of vehicle or JMV2959 ( 10 μg ) and having their food intake recorded ( as described above ) . A between-subjects design was used to avoid conditioning effects with repeated food restriction . Given the baseline differences in food intake between meal-entrained and control rats , to control for a lack of observable effects based on low kcal intake ( i . e . , floor effect ) in controls , a separate cohort of non-food restricted rats ( n=11 ) were first habituated to 1 ~3 g pellet of a ‘Western diet’ a day ( 45% kcal fat , enriched with sucrose; Research Diets D12451 ) for 5 days , given at random times to avoid conditioning effects . Two days later , animals received vHP injections of 0 µg , 1 µg , or 5 µg JMV2959 ( total doses: 0 µg , 2 µg , 10 µg ) using a counter-balanced within subjects design ( treatments separated by 2–3 intervening days ) and food intake was recorded as described above . To determine whether endogenous LHA ghrelin signaling also contributes to conditioned feeding responses , we repeated the meal entrainment ( n=9 ) and non-entrained , 20 hr food deprivation ( n=15 ) procedures described above with bilateral , intra-LHA injections of JMV2959 ( total doses: 0 μg , 2 μg , 10 μg ) . Each rat ( n=6 ) received a unilateral injection of CTB and a unilateral contralateral injection of FG targeted to the dorsal perifornical LHA ( dpLHA ) . Seven days later , rats were fixation-perfused , and brains removed and processed as described . Fluorogold was examined using its native fluorescence . Detection of CTB by IHC followed the steps described above , except the following antibodies and fluorophores were used: anti-CTB primary antibody ( mouse monoclonal , 1:500 , Abcam , Cambridge , MA Catalog #ab62429 ) ; biotinylated anti-mouse secondary antibody ( 1:500 , donkey polyclonal , Jackson Immunoresearch , West Grove , PA ) ; streptavidin DyLight 549 tertiary reagent ( 1:500 , Jackson Immunoresearch; overnight ) . For IHC detection of GHSR the following antibodies were used: Anti-GHSR1 primary antibody ( goat , polyclonal; 1:500 , Santa Cruz , Santa Cruz , CA , Catalog #sc-10362 ) ; anti-goat Alexa Fluor 488 secondary antibody ( donkey polyclonal , 1:500 , Jackson Immunoresearch ) . Rats received unilateral NMDA injections targeted to the dpLHA ( n=10 ) or sham lesions ( n=7 ) and cannulae targeting the bilateral vHP . 2 hr into the rats light cycle ( lights on at 8 AM ) , 0 pmol or 300 pmol ghrelin was administered to the vHP ipislateral to the dpLHA lesion . Unilateral ghrelin doses were determined based on pilot studies ( data not shown ) and previous literature ( Kanoski et al . , 2013 ) , where the maximally effective dose for hyperphagia was chosen . In a separate experiment , the same cohort received 0 pmol or 300 pmol ghrelin to the vHP contralateral to the dpLHA lesion . Treatments were separated by intervening 1 day using a counterbalanced mixed design , with sham or lesion as a between-subjects variable , and drug as within-subjects . Chow intake was recorded at 1 , 3 , and 5 hr after injections ( food spillage accounted for ) . Rats ( n = 4 ) received iontophoretic injections of PHAL targeting the vHP . Seven days later , the rats were euthanized and tissue was harvested and processed according to procedures outlined in Materials and methods . IHC detection of PHAL proceeded according to the above described procedures using goat anti-PHAL primary antibody ( 1:5000 , Vector Labs , Catalog #AS-2224 ) , followed by a donkey anti-goat secondary antibody conjugated to Cy3 ( 1:500 , Jackson Immunoresearch , 4 hr ) . Material from the experiment with a PHAL injection site that was most restricted to the vHP field CA1 region of interest was selected for subsequent IHC analysis of vHP connection interactions with dpLHA orexin neurons . IHC detection of orexin was performed as described using a rabbit anti-ORX1 primary antibody ( 1:1000 , Phoenix Pharmaceuticals , Burlingame , CA; Catalog #H-003-30 , 18 hr ) , was followed bya donkey anti-rabbit secondary antibody conjugated to AMCA ( 1:500 , Jackson Immunoresearch , ) . IHC detection of synaptophysin was performed as described using a mouse anti-synaptophysin primary antibody ( 1:1000 , PROGEN Biotechnik , Heidelberg , Germany; Catalog #61012 , 18 hr in room temperature ) , followed by a donkey anti-mouse secondary antibody conjugated to Alexa Fluor 488 ( 1:500 , Jackson Immunoresearch ) . Rats received either unilateral vHP ghrelin ( n=5; 300 pmol ) or aCSF vehicle injections ( n=4 ) . 60 min later , animals were euthanized and tissue was harvested and processed according to procedures outlined in Materials and methods . Fos was used as a marker for neuronal activity , and IHC detection was performed according to the procedure above using a sheep anti-Fos primary antibody ( 1:2000 , Cell Signaling , Danvers , MA; Catalog # 2250s , 18 hr ) , followed by a donkey anti-goat secondary antibody conjugated to Alexa Fluor 488 ( 1:500 , Jackson Immunoresearch , 4 hr ) . IHC detection of orexin was performed as above using a rabbit anti-Ox1a primary antibody ( 1:1000 , Phoenix Pharmaceuticals , Catalog #H-003-30 , 18 hr ) and was followed up with an incubation of a donkey anti-rabbit secondary antibody conjugated to Cy3 ( 1:500 , Jackson Immunoresearch ) . Quantification of co-localization of Fos and orexin positive cells was calculated by an experimenter blind to the treatment conditions using ImageJ software ( cell count Plugin ) based on the number of co-localized Fos and orexin cells divided by the total number of orexin positive cells . Rats ( n=15 ) received cannulae targeting the both the vHP and LV . 2 hr into light cycle , animals received LV injections of 0 nmol or 30 nmol the orexin-1 receptor antagonist , SB334867 , immediately followed by 0 pmol or 300 pmol ghrelin to the vHP . Dose of SB334867 was determined based on previous literature ( Parise et al . , 2011; Haynes et al . , 2000 ) . Treatments were separated by 1 intervening day using a counterbalanced within-subjects design . Chow intake was measured 1 , 3 , and 5 hr after injections ( food spillage accounted for ) . All statistical analyses employed repeated measures or one-way analysis of variance ( ANOVA ) . The α level for significance was 0 . 05 . Statistical analyses were conducted with computer software ( Statistica V7; Statsoft ) . To ensure the minimum sample size required to be reasonably likely to detect the hypothesized effect , power analyses were conducted for each proposed experiment based on our pilot and recently published experiments using Statsoft software ( Statistica 64 , V7 ) . Alpha level was set at 0 . 05 for power analyses; the Root Mean Square Standardized Effect ( RMSSE ) was estimated separately for each experiment
Feeding behavior rarely occurs in direct response to metabolic deficit , yet the overwhelming majority of research on the biology of food intake control has focused on basic metabolic and homeostatic neurobiological substrates . Most animals , including humans , have habitual feeding patterns in which meals are consumed based on learned and/or environmental factors . Here we illuminate a novel neural system regulating higher-order aspects of feeding through which the gut-derived hormone ghrelin communicates with ventral hippocampus ( vHP ) neurons to stimulate meal-entrained conditioned appetite . Additional results show that the lateral hypothalamus ( LHA ) is a critical downstream substrate for vHP ghrelin-mediated hyperphagia and that vHP ghrelin activated neurons communicate directly with neurons in the LHA that express the neuropeptide , orexin . Furthermore , activation of downstream orexin-1 receptors is required for vHP ghrelin-mediated hyperphagia . These findings reveal novel neurobiological circuitry regulating appetite through which ghrelin signaling in hippocampal neurons engages LHA orexin signaling .
Eating can occur for many different reasons . We often eat not simply because we are hungry , but because we have learned to eat at specific times of day or in response to factors in our environments ( for example , an advert that reminds of us of something tasty ) . Despite this , few researchers have investigated how biological signals from the gut communicate with the brain to enable this type of learned , or “conditioned” , feeding behavior . Brain cells called neurons communicate with each other in specific circuits that can cross between different brain regions . Hsu et al . have now developed a “disconnection neuropharmacology” approach , which alters whether the neurons in different brain regions can communicate with each other , to investigate the effects signaling molecules have on brain activity and behavior . Using this approach in rats revealed a new circuit in the brain that controls learned feeding behavior through ghrelin , a hormone that is released from the stomach and increases appetite . Hsu et al . found that the hippocampus , a brain region important for learning and memory control , uses ghrelin as a signal to engage in learned feeding behavior . Neurons in the hippocampus that respond to ghrelin communicate with other neurons in a region of the brain called the lateral hypothalamus ( known for its role in feeding ) that produce a signaling molecule called orexin . This circuit therefore links memory and feeding behavior . Future work will investigate the neural circuits between the hippocampus and other brain regions that are important for feeding behavior , and the hormones that are important for triggering activity in these circuits .