Ghrelin: a Link Between Memory and Ingestive Behavior

PHB-11276; No of Pages 8 Physiology & Behavior xxx (2016) xxx–xxx

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Physiology & Behavior

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Review Ghrelin: A link between memory and ingestive behavior

Ted M. Hsu a,b, Andrea N. Suarez a, Scott E. Kanoski a,b,⁎ a Human and Evolutionary Biology Section, Department of Biological Sciences, University of Southern California, Los Angeles, CA, USA b Neuroscience Program, University of Southern California, Los Angeles, CA, USA

HIGHLIGHTS

• Learning and memory processes strongly inﬂuence feeding behavior. • Learned, cephalic endocrine responses are a physiological substrate for conditioned feeding. • The cephalic ghrelin response is a learned, anticipatory signal for feeding. • Ghrelin acts in hippocampus-lateral hypothalamic neural circuitry to express learned feeding.

article info abstract

Article history: Feeding is a highly complex behavior that is inﬂuenced by learned associations between external and internal Received 13 January 2016 cues. The type of excessive feeding behavior contributing to obesity onset and metabolic deﬁcit may be based, Received in revised form 29 March 2016 in part, on conditioned appetitive and ingestive behaviors that occur in response to environmental and/or inter- Accepted 31 March 2016 oceptive cues associated with palatable food. Therefore, there is a critical need to understand the neurobiology Available online xxxx underlying learned aspects of feeding behavior. The stomach-derived “hunger” hormone, ghrelin, stimulates appetite and food intake and may function as an important biological substrate linking mnemonic processes Keywords: Appetite with feeding control. The current review highlights data supporting a role for ghrelin in mediating the cognitive Learning and neurobiological mechanisms that underlie conditioned feeding behavior. We discuss the role of learning and Obesity memory on food intake control (with a particular focus on hippocampal-dependent memory processes) and pro- Hippocampus vide an overview of conditioned cephalic endocrine responses. A neurobiological framework is provided through GHSR which conditioned cephalic ghrelin secretion signals in neurons in the hippocampus, which then engage Cephalic orexigenic neural circuitry in the lateral hypothalamus to express learned feeding behavior. © 2016 Elsevier Inc. All rights reserved.

Contents

1. Introduction...... 0 2. Mnemoniccontroloffoodintake...... 0 3. Learnedcephalic-phasebiologicalresponsesandfeedingbehavior...... 0 4. Ghrelin:acephalicsignalfortheanticipationoffeeding...... 0 5. CNSregulation:hippocampalcontrolofghrelin-mediatedconditionedfoodintake...... 0 6. Conclusions...... 0 Acknowledgements...... 0 References...... 0

1. Introduction of feeding behavior, in particular the type of excessive food intake that contributes to weight gain and metabolic disorders. Food seeking and In light of the current obesity pandemic, there is a recent surge of ingestion rarely occur as passive behaviors in response to energy deﬁcit. interest in investigating the biological and psychological antecedents Rather, for humans, food intake is more often initiated as a result of either habitual feeding patterns (e.g., meals consumed at approximately ⁎ Corresponding author at: University of Southern California, 3616 Trousdale Parkway, AHF-252, Los Angeles, CA 90089-0372, USA. the same time each day) or in response to environmental stimuli asso- E-mail address: [email protected] (S.E. Kanoski). ciated with food consumption, including cues associated with palatable

Please cite this article as: T.M. Hsu, et al., Ghrelin: A link between memory and ingestive behavior, Physiol Behav (2016), http://dx.doi.org/ 10.1016/j.physbeh.2016.03.039 2 T.M. Hsu et al. / Physiology & Behavior xxx (2016) xxx–xxx foods such as fast food billboards and TV commercials. Indeed, learned recollection of factual information (semantic memory) and autobiograph- associations between food and interoceptive or external cues have the ical memories (episodic memory), requires neural processing in the hip- capability to drive or alter feeding behavior [1–5]. However, despite pocampus [22,23]. Declarative memories of previous meals or eating the robust influence of mnemonic processes on food intake, very little occasions can be consolidated and retrieved at a later time to influence is understood about the neurobiological substrates of learned feeding subsequent feeding behaviors. This is perhaps most powerfully illustrated behavior. In this review we discuss psychological and biological mecha- by research in amnesic patients with bilateral damage to the hippocam- nisms underlying learned feeding behavior, focusing primarily on the pus and surrounding medial temporal lobe. These patients are impaired gut-derived hormone, ghrelin as being a critical interface linking mem- in episodic memory formation, and under some experimental conditions ory and ingestion. they will consume a second or third meal offered only minutes after con- suming a meal [24,25]. Parent and colleagues [26] have elegantly modeled 2. Mnemonic control of food intake this phenomenon in rodent models, demonstrating that reversible inacti- vation of hippocampal neurons immediately following a scheduled, antic- Learning and memory processes have powerful control over appe- ipated sucrose meal reduces meal initiation latency and increases the size tite and food intake. Through experience, food-associated cues (e.g. of the subsequent meal [26]. This group more recently demonstrated that environmental cues or interoceptive cues) become associated with orosensory stimulation through either intake of sucrose or non-caloric rewarding or aversive postingestive consequences, and these learned saccharin robustly increases hippocampal synaptic plasticity in an associations robustly affect future feeding behaviors. An example is experience-dependent manner [27]. Together, these results suggest conditioned flavor avoidance (or aversion) learning, a process through that 1) hippocampal neural processing can regulate the consolidation which animals learn to avoid (or reject) neutral flavors that were previ- of food-related declarative memories, and 2) disrupted hippocampal ously associated with nausea or malaise [6]. Studies have also dem- processing can potentially lead to excessive eating. Indeed, rodents with onstrated appetite promoting effects through learned associations selective bilateral hippocampal lesions are hyperphagic and gain excess between neutral flavor cues and nutritive postingestive consequences. body weight relative to control rats [28]. More specifically, animals can learn to prefer a neutral flavor that was pre- An alternative and complementary framework for the influence of viously paired with gastric or intestinal infusion of nutrients [7].Thus, hippocampal-dependent memory processes on feeding behavior – one even simple associative learning mechanisms have the capacity to power- that does not evoke the use of complex mnemonic constructs like fully modulate feeding either by stimulating appetite or by inducing declarative memory – is provided by Davidson and colleagues (see avoidance/aversive behaviors. This review will focus on the role of ghrel- [3,28–31] for reviews on the role of hippocampal neural processing in, an appetite-promoting hormone released primarily from the gastric P/ in the mnemonic regulation of feeding behavior). In this model, D1 cells, on learned aspects of eating behavior. hippocampal-dependent memory processes influence feeding by In addition to associations between food-relevant stimuli and utilizing interoceptive energy status cues (e.g., different levels of food postingestive consequences, learned relationships between food access restriction) to resolve ambiguous learned relationships between food- and other types of interoceptive cues, including circadian rhythms, also related stimuli and their postingestive consequences [32]. Within this have the capacity to guide and alter feeding behavior. Many humans framework, energy status cues (e.g., hunger, satiety) modulate the and nonhuman animals have habitual feeding patterns in which meals strength of learned associations between food-related stimuli and are consumed at approximately the same time each day (e.g., breakfast, postingestive nutritive reinforcement. These energy status cues are likely lunch, and dinner), and accordingly, conditioned interoceptive circadian communicated to the hippocampus, in part, by ghrelin and other endo- cues can acquire the capacity to elicit preparatory cephalic release of crine signals (e.g., leptin, glucagon-like peptide-1) whose receptors are ghrelin and other endocrine signals that influence appetitive behavior expressed on hippocampal neurons [33]. [8–10] (a phenomenon discussed in more depth below). In some cases Based on the work described above, it is apparent that both simple feeding behavior is entrained, not based on convenient habitual feeding learned associations and more complex hippocampal-dependent mne- patterns, but rather, based on limited food availability. This type of monic processes are critical for regulating decisions about when and entrainment can be demonstrated experimentally in rodents placed on where to feed, as well as preparing an organism to consume food when restricted meal schedules in which they are only allowed access to food feeding is imminent or anticipated. Regarding the latter, sensory stimuli at a fixed time and duration each day. Animals under these feeding condi- associated with meal anticipation elicit preparatory physiological “ce- tions learn across days to rapidly consume a large quantity of food to meet phalic” reflexes that occur prior to feeding [34–38]. These responses are their metabolic requirements [10–12]. particularly advantageous when environmental factors, such as limited Food-associated environmental cues are also capable of driving hyper- food availability or access, restrict food consumption such that an organ- phagia under conditions where food access is not limited or restricted. ism is required to consume a large amount of food in a short period of Previously neutral cues (e.g. visual or auditory stimuli) that become asso- time. Research has demonstrated that circadian, olfactory, gustatory, ciated with access to palatable food can potently stimulate appetite and and cognitive food-related cues are capable of generating these cephalic excessive feeding behavior (i.e., “cue potentiated feeding”) in both phase responses, characterized by transient upregulation of key endo- humans and nonhuman animal models. For example, rodents can learn crine feeding signals such as ghrelin and the pancreatic hormone insulin to associate discrete auditory stimuli with rewarding foods, and subse- [10,34,38]. The integration of ghrelin and other cephalic-released hor- quent exposure to the food-associated cue(s) when the same food rein- mones with higher-order neural substrates that control learning and forcement is freely available stimulates intake in nonrestricted rats in memory processes may represent a mechanistic biological link between excess of that occurring during or after exposure to control cues [1,2,5, food-related mnemonics, interoceptive energy status cues, and the neural 13–17]. In fully sated humans, the sight of palatable foods enhances re- control of ingestive behavior. ported desire and intake for the given food [1], and exposure to recogniz- able food advertisements and other food cues increases subsequent food 3. Learned cephalic-phase biological responses and feeding behavior intake in children [5,18]. Importantly, the processes through which food- associated external cues promote excess palatable food consumption in- Cephalic-phase responses are anticipatory physiological and metabol- volve ghrelin signaling [19–21]. ic adjustments in preparation for the digestion and absorption of ingested In addition to influencing feeding through simple associations and nutrients and reflect a biological mechanism through which food cues in- learned circadian entrainment patterns, food-related conditioned cues teract with the central nervous system to express learned feeding behav- can influence food intake through their integration into more complex ior. Nutrient ingestion, while obviously beneficial, can also be thought of mnemonic constructs. Declarative memory, comprised of the conscious as perturbing physiological homeostasis. By eliciting cephalic-phase

Please cite this article as: T.M. Hsu, et al., Ghrelin: A link between memory and ingestive behavior, Physiol Behav (2016), http://dx.doi.org/ 10.1016/j.physbeh.2016.03.039 T.M. Hsu et al. / Physiology & Behavior xxx (2016) xxx–xxx 3 responses, disruption of homeostasis during feeding is attenuated and In addition to insulin, there are several other feeding relevant hor- food intake during a meal is enhanced (reviewed in [39]). For example, monal cues that have the capacity for conditioned cephalic phase rats that are placed on a restricted feeding schedule involving random release. For example, circulating glucagon-like peptide-1 (GLP-1) levels meal times have been shown to eat smaller meals compared to ad have been shown to increase prior to anticipated meals in rodents [9, libitum-fed rats [39]. In addition to circadian entrainment cues, exposure 52]. Studies have also suggested possible cephalic leptin responses, as to sensory related food cues (e.g., orosensory, visual) and cognitive factors sham-feeding procedures augment circulating leptin levels [53] and (e.g., thinking about food) can generate cephalic-phase responses leptin levels are regulated by circadian rhythms, with robust prandial (discussed below). These cephalic-phase responses involve Pavlovian peaks in scheduled-fed humans and rodents [54–56]. However, experi- conditioning principles in the sense that animals learn through experiments examining the cephalic leptin response have been inconsistent ence that a certain food cue occurs prior to or during feeding (uncondi- and require further research. Glucagon also has a potential cephalic tioned stimulus), and that this food cue subsequently acts as a role as experiments have demonstrated that levels of glucagon decrease conditioned stimulus that acquires the capacity to evoke conditioned rein the hours preceding an anticipated meal in rats [57], an effect in the sponses; the cephalic-phase responses. Indeed, a classic example of a con- opposite direction of most other cephalic-phase endocrine responses. ditioned cephalic response is salivation; an effect famously demonstrated Evidence for cephalic pancreatic polypeptide (PP) release has also in dogs in Pavlov's pioneering work in classical conditioning theory. been demonstrated through robust increases in circulating PP levels The most extensively studied cephalic-phase response is cephalic in- during sham feeding [58–60]. Collectively, these studies and others sulin release. In humans, preprandial insulin infusion significantly reduces [36,61,62] have begun to elucidate the physiological and endocrine postprandial glucose levels [40], thereby supporting an important role of mechanisms that mediate learned food intake. Below we focus on the preabsorptive insulin release contributing to meal related glucose homeo- gut-derived “hunger” hormone ghrelin as a particularly important link stasis. The vagus nerve (10th cranial nerve), which relays gastrointestinal between conditioned cephalic-phase endocrine responses and neural food-related sensory signaling to the brain, plays a critical role in cephalic regulation of feeding behavior. insulin release. Vagal lesions in sheep decrease the insulin response after presentation of food and increase postprandial glucose levels [41]. Simi- 4. Ghrelin: a cephalic signal for the anticipation of feeding larly, truncal vagotomy has been shown to disrupt cephalic insulin release in rats [42]. Ghrelin is the only known circulating hormone that increases food In addition to insulin secretion, gastric emptying is another intake [63,64] and has prominent stimulatory roles in modulating vagally-mediated cephalic-phase response. Myoelectric activity of meal size, meal frequency, body weight, and food-motivated reward be- the stomach, which correlates to gastric motility, increases in sham haviors ([21,65–67]; see reviews [68,69]). While ghrelin is commonly fed humans (sight, smell, and taste of food without ingestion) but referred to as a “hunger” hormone, a growing body of literature in not in vagotomized individuals [43]. Similarly, sham feeding im- both human and animal models indicates that ghrelin is more appropri- proves gastric motility in humans with impaired gastric digestion ately described as a signal for the anticipation of food intake. For exam- [44].Thesefindings suggest that orosensory food-related cues, com- ple, rodents who learn to anticipate meals when placed on a meal monly modeled experimentally with sham feeding, affect vagus entrainment schedule (e.g., fixed 4 h food access every day at the nerve signaling to elicit cephalic-phase responses such as cephalic same time) exhibit increasing levels of circulating ghrelin prior to the insulin release and gastric motility. ingestion of the meal, followed by rapid postprandial suppression. Im- Olfactory and orosensory food-related cues are perhaps the most ro- portantly, levels of ghrelin measured immediately prior to the learned bust stimulators of cephalic-phase responses. In humans, increased insu- food access period were elevated in comparison to animals that were lin secretion is elicited within 2 min after orosensory stimulation (without equally food-restricted but had not received previous meal entrainment ingestion) and returns to baseline levels 10 min after the stimulus was ini- conditioning [10]. Comparable results are also found in sheep that were tiated [36]. Similarly, in the rat, continuous sham feeding via gastric fistu- fed once daily at the same time each day, where experimenters showed las produces robust cephalic insulin release and this response is blocked that immediately prior to the animals' meal, there is a transient surge of when vagal signaling is disrupted with atropine administration [45].In plasma ghrelin levels that rapidly declines during feeding. Interestingly, addition to the robust cephalic-phase responses produced from this study also showed a similar cephalic ghrelin response to “pseudo- orosensory and olfactory cues, exposure to visual food-related cues also feeding”, where the animals were given the diet in a nylon bag that influences cephalic-phase responses. Forexample,indogs,gastricacidse- could not be ingested [70]. Similarly, in humans who are instructed to cretion occurs following the sight of food, however, this response is less anticipate a large meal following a 14-hour fast and then exposed to robust than that occurring following sham feeding via esophageal fistula the presentation of a breakfast buffet, a rapid postprandial suppression stimulation [46]. Presentation of pictures of highly palatable foods also of plasma ghrelin levels occurs that is in excess of that observed in elicits an increase in salivation compared to normal, resting salivary levels equally fasted-controls [71]. While the study showed no differences in in both rats [47] and humans [48]. In this latter study, subjects that were preprandial ghrelin levels compared to equally fasted-controls (poten- able to consume the food that was viewed compared to those that were tially caused by knowledge of future food intake in control subjects), told they couldn't consume food showed higher salivary responses [48], the results suggest that meal anticipation can strongly modulate periph- suggesting that under some conditions cephalic-phase responses are eral ghrelin secretion in humans. also under cognitive control. In further support of ghrelin as an anticipatory signal for food intake, Circadian cues have also been demonstrated to initiate cephalic in- Cummings and colleagues [72] measured plasma ghrelin levels in sulin secretion, thereby allowing an animal to exhibit conditioned phys- humans with meals provided on a fixed schedule throughout a 24- iological changes at consistent time points with relation to habitual or hour period. Their results revealed a robust increase in ghrelin levels im- restricted feeding schedules. Rats placed on a restricted feeding sched- mediately before each scheduled meal (breakfast, lunch, and dinner), ule of food access for 2 h everyday secrete large levels of insulin at the followed by a suppression occurring 1 h afterwards [72]. Other studies time of day associated with feeding (even without exposure to the using human subjects have also demonstrated that varying habitual food) compared to rats with ad libitum access to food [8,49]. At a cellular feeding times (e.g., short or long inter-meal intervals) strongly affects level, transcription factors CLOCK and BMAL1 are circadian “clock temporal patterns of circulating ghrelin concentrations, where peak genes” expressed in the beta cells of the pancreas, and their functional ghrelin levels occur immediately prior to an individual's habitual meal elimination in mutant mice results in decreased insulin production time [73]. These results suggest that ghrelin levels rise in anticipation and high levels of blood glucose [50,51]. Thus, cephalic insulin release of food intake based on learned, habitual feeding patterns as opposed based on circadian cues may be due to entrainment of these clock genes. to being exclusively a “hunger signal” whose release is linked to the

Please cite this article as: T.M. Hsu, et al., Ghrelin: A link between memory and ingestive behavior, Physiol Behav (2016), http://dx.doi.org/ 10.1016/j.physbeh.2016.03.039 4 T.M. Hsu et al. / Physiology & Behavior xxx (2016) xxx–xxx magnitude of food restriction. Scheduled feeding regiments (3 meals a the vagus required for postprandial suppression of ghrelin [87].Howev- day at fixed times) in rodents also induce spontaneous activity and in- er, total subdiaphragmatic vagotomy eliminated the hyperphagic effects creased acylated ghrelin levels at the time of scheduled meal presenta- of intravenous ghrelin administration in rats [88], and the increases in tion [74]. Collectively, these data suggest that ghrelin acts as an circulating ghrelin in response to 48 h food restriction [85]. These anticipatory signal for meal initiation, and in these examples its release mixed findings give little clear insight as to whether cephalic phase is potentially stimulated, in part, by learned circadian cues. This notion ghrelin release operates independent of the vagus, and to our knowl- is further corroborated by studies demonstrating that stomach cells edge this has not yet been directly tested. Regardless, it is clear that that secrete ghrelin also express circadian clock proteins (PER1 and learned processes involving food access (or eating) can entrain the se- PER2) that exhibit rhythmic activity in response to food entrainment cretion of ghrelin, which may act as an important signal for meal initia- [75]. Moreover, in one study ghrelin levels increased before requested tion and anticipatory feeding behavior through activity within the meals in subjects that were deprived of time- and food-related cues central nervous system. [76], suggesting a circadian entrainment. However, an alternative (and The data reviewed above provides strong evidence for the role of ce- not mutually exclusive) explanation of these findings is that ghrelin phalic ghrelin signaling in meal anticipation, and together with activa- levels rise with hunger rather than (or in addition to) in response to cir- tion of ghrelin receptors (growth hormone secretagogue receptor, cadian rhythms. GHSR), represents a biological signaling system that allows for learned Other food related stimuli are also capable of generating cephalic feeding behavior. Previous data has shown that genetic deletion of ghrelin responses. In humans undergoing a modified sham-feeding par- GHSR results in deficits in meal anticipatory behavior under meal en- adigm, serum ghrelin levels exhibit a typical cephalic response in which trainment schedules ([89–91], but see [92]) and as indicated above, there is a rise immediately following sham feeding, followed by rapid both pharmacological blockade and genetic knockout of GHSRs disrupt suppression that in some cases can occur despite the lack of actual in- the expression of cue potentiated feeding [19,20]. These findings sug- gestion [34,77]. These findings suggest that ghrelin release triggered gest that not only is ghrelin receptor signaling important for the expres- by orosensory food cues can return to baseline without postingestive sion of learned feeding behavior based on internal cues (e.g., circadian), nutritive consequences, potentially through a yet unidentified cephalic but also for feeding driven by external, food-associated stimuli. Interest- phase response contributing to the clearance of ghrelin from circulation. ingly, Davis and colleagues [89] showed that GHSR null mice can meet Another study revealed an increase in circulating ghrelin levels occurred metabolic requirements under meal-entrained schedules, but are im- following sham feeding and this response was markedly higher in indi- paired in meal anticipatory locomotor activity. While these data suggest viduals with anorexia than age-matched healthy controls [78]. Similar- that ghrelin signaling is not required for food intake control in response ly, research from the same group reported that in women with bulimia to predictable metabolic deficits, we note that compensatory mecha- there is a higher cephalic increase in ghrelin levels relative to healthy nisms are likely to arise during development as a result of genetic dele- controls in response to sham feeding, a result that is positively correlat- tion of GHSRs. Thus, conclusions with regards to the ghrelin's functional ed with binge eating frequency [79]. These results taken together sug- relevance to meal anticipation (and to learned consummatory re- gest that orosensory stimulation can have a powerful affect on sponses) that are made exclusively based on transgenic GHSR null circulating ghrelin levels, however, more research is needed to resolve mice should be interpreted with caution. It should also be noted that the temporal dynamics and other factors contributing to ghrelin secre- the preceding studies did not systematically determine whether trans- tion vs. clearance immediately following orosensory stimulation. genic or pharmacological-induced deficits in ghrelin signaling impair The cephalic-phase ghrelin response is also differentially regulated distinct associative processes related to food cues (e.g., acquisition, con- depending on macronutrient content. Zhu and colleagues [80] demon- solidation, and retrieval), nor do they identify a specificneuronalsub- strated that modified sham feeding of different types of diet (e.g. strate that potentially regulates ghrelin's effects on learned feeding high-fat, carbohydrate, or protein) all induce a cephalic ghrelin re- behavior. sponse, however, there is a substantially greater increase in circulating ghrelin levels with high-protein foods. These data indicate that the ce- 5. CNS regulation: hippocampal control of ghrelin-mediated phalic ghrelin response is not only sensitive to previous experience, conditioned food intake but are also flexible to changes in macronutrient composition and/or the type of diet of that is being consumed. Whether palatability and/or Ghrelin readily crosses the blood brain barrier [84,93] and acts flavor of the diet modulate ghrelin release independently of macronu- through the G protein-coupled receptor, GHSR. GHSRs are densely trient content will require further investigation. expressed in many feeding and reward-related neural substrates [94, While these studies strongly implicate ghrelin as an anticipatory 95] and studies have shown that ghrelin acts within the limbic and feeding signal released in response to conditioned circadian and/or mesolimbic reward systems [e.g., ventral tegmental area (VTA), nucleus orosensory cues, it is not yet known whether cephalic ghrelin responses accumbens (NAcc) and amygdala] to express a myriad of ingestive and occur in response to visual and other food cues (e.g., cognitive, intero- food-motivated behaviors [96–100]. For example, direct ghrelin admin- ceptive). However, as indicated above ghrelin signaling has an impor- istration to either the VTA or NAcc increases food intake, whereas moti- tant role in external, cue-driven feeding. For example, peripheral vated responding for food reward is increased following VTA (but not ghrelin receptor blockade [19] or genetic deletion [20] in rodents blocks NAcc) ghrelin delivery [98,99]. the ability of Pavlovian conditioned stimuli to produce hyperphagia. The ventral subregion of the hippocampus (vHP) has also been Whether these types of external cues generate cephalic ghrelin re- recently identified as playing a role in food-motivated and ingestive be- sponses requires deeper investigation. Moreover, the role of cephal- haviors [3,28–31,33]. The hippocampus is activated by gastric disten- ic ghrelin secretion in hyperphagia and obesity generally speaking is sion and nutrient infusion [101,102] and has been implicated in the not established. Several studies have shown that obese humans [81, modulation of learned meal anticipation [103–106]. Neurons in the 82] and rodents maintained on an obesogenic (e.g., high fat) diet ex- vHP express receptors for various endocrine feeding-relevant signals, hibit impaired ghrelin secretion [83–85] and central ghrelin signal- including leptin, GLP-1, and ghrelin [94,107,108], and activation of ing [21]. Future systematic studies will be required to determine these receptors in the vHP is one mechanism through which hippocam- whether impairments in cephalic ghrelin signaling contribute to pal neural processing bi-directionally influences energy balance [21, obesity development. 109,110]. Interactions between these vHP neuroendocrine signals are It is also unclear whether cephalic ghrelin responses require vagal a strong possibility and a deeper discussion on this topic can be found signaling. Vagal afferents are not required for the hyperphagic effects in [33]. Recent work from Kanoski, Grill, and colleagues [21] showed following peripheral (intraperitoneal) ghrelin injections [86], nor is that pharmacological activation of GHSRs in the vHP potently increases

Please cite this article as: T.M. Hsu, et al., Ghrelin: A link between memory and ingestive behavior, Physiol Behav (2016), http://dx.doi.org/ 10.1016/j.physbeh.2016.03.039 T.M. Hsu et al. / Physiology & Behavior xxx (2016) xxx–xxx 5 food intake and food motivated behaviors. They further demonstrated orexin signaling) through direct action on LHA orexin neurons. However, that vHP GHSR activation increases meal initiation frequency in re- blockade of GHSRs in the LHA has no effects on food intake in meal- sponse to external food-associated cues, while having no hyperphagic entrained animals [111], indicating that the vHP ghrelin signaling is a po- feeding effects in response to neutral stimuli [21]. This is strong evi- tentially a unique neural substrate for learned feeding behavior. dence for vHP ghrelin signaling as a neural substrate that underlies Taken together, these data suggest that through monosynaptic neu- learned feeding behavior in response to external food-associated cues. ral circuitry between the vHP and LHA, vHP ghrelin-activated gluta- To expand these findings, a recent study from our group investigated matergic pyramidal CA1 neurons stimulate LHA orexin secretion, the endogenous relevance of this system in circadian entrained feeding which in turn engages the expression of learned feeding through ac- behavior [111]. Rats were placed on a meal entrainment schedule in tivation of orexin receptors in downstream regions, potentially in- which food was presented at the same time every day for 4 h. Similar to cluding the VTA. However, in addition to the VTA, orexin receptor previous work [10], these animals learned to utilize internal circadian signaling at several brain regions has diverse effects on feeding cues to anticipate meals, and steadily increased their caloric intake over and food-motivated behaviors [119,130,132,133,138–141].Thus, successive days to meet their metabolic needs. Pharmacological blockade there is a need for future studies to explore which of the various po- of vHP GHSRs (via JMV 2959, a GHSR antagonist) prior to meal access re- tentialdownstreamorexintargetsare relevant for learned feeding duced 4-hour food consumption during the scheduled meal time, while effects. Furthermore, vHP field CA1 neurons also have projections having no effects in animals that were similarly food deprived, but not to other feeding relevant regions, such as the amygdala and medial meal entrained. These data strongly suggest that vHP ghrelin signaling prefrontal cortex (mPFC) [142,143], which then provides topo- is a neurobiological substrate that is endogenously relevant for learned graphical input to the LHA [115,116]. Additionally, amygdala-LHA food intake. It is also important to note that GHSRs have high levels of and mPFC-LHA neural pathways are required for the expression of constitutive activity, and there is emerging research suggesting that con- cue potentiated feeding [13,15]. Whether these polysynaptic path- stitutively active GHSRs might play a role in regulating energy balance ways are also involved in vHP ghrelin-mediated feeding effects [112,113]. Thus, while ghrelin release and vHP ghrelin signaling is impor- will require deeper examination. tant for learned feeding behavior, there is the yet unexplored possibility Based on our recent work and other previous findings, we propose that vHP GHSR constitutive activity is also involved in regulating meal an- the following model through which ghrelin acts to regulate conditioned ticipation and subsequent food intake control. This hypothesis could be aspects of feeding (Fig. 1;modified from [111]): A) meal anticipation is 1 5 7,9 tested using a GHSR inverse agonist (e.g., [D-Arg , D-Phe , D-Trp , induced by learned external or internal food associated cues, which Leu11]-substance P [114]) to silence constitutive activity in vHP GHSR- B) stimulates the secretion of ghrelin from the P/D1 cells in the stomach. expressing neurons. C) Ghrelin then enters the vascular system, where it crosses the blood- Field CA1 pyramidal neurons in the vHP ipsilaterally project to the brain barrier and accesses the brain. D) Ghrelin activates vHP field CA1 dorsal perifornical subregion of the lateral hypothalamic area (dpLHA) pyramidal neurons, which project monosynaptically to LHA orexin neu- [111,115–118], a region that mediates orexigenic aspects of feeding be- rons. E) The activated vCA1 pyramidal neurons then stimulate orexin- havior [119–126].Hsuetal.[111] demonstrated that ~85% of vCA1 neu- producing LHA neurons and initiates downstream orexin signaling to rons that project to the dpLHA also express GHSR [111], thus providing feeding and reward-relevant brain regions (e.g. VTA). F) Activity within anatomical evidence identifying the LHA as a downstream target for this system then stimulates learned appetite and food intake. vHP ghrelin signaling. Taking advantage of the exclusively ipsilateral connection between these brain regions, a neuropharmacological neural dis- 6. Conclusions connection approach was utilized that eliminated communication between the vHP and dpLHA via unilateral excitotoxic lesions of the Feeding is a highly complex behavior and several studies have now dpLHA. In unilateral dpLHA-lesioned and sham control rats, food intake shown an intimate relationship between learning and memory process- was measured following unilateral injections of ghrelin either ipsilateral es and their underlying neural and physiological substrates in the con- (vHP-dpLHA connection compromised) or contralateral (vHP-dpLHA trol of energy balance. The gut-derived hormone ghrelin is a biological connection intact) to the dpLHA lesion. A hyperphagic response was ob- signal that appears to be a critical link between previous experience served when ghrelin was administered to the vHP contralateral to the and food intake control. Like insulin and other endocrine signals, ghrelin dpLHA lesions, and in animals with sham lesions; however, when ghrelin is released in a cephalic manner based on learned meal entrainment and was administered ipsilaterally to the dpLHA lesion the hyperphagic re- orosensory stimulation, and may also be released in response to other in- sponse was robustly attenuated [111]. These data strongly suggest that teroceptive and external food-related cues. Circulating ghrelin crosses the the LHA is a critical downstream target for vHP ghrelin-mediated feeding blood-brain barrier and acts in several brain regions to stimulate appetite and therefore highlight a novel neural circuitry mediating some aspects of and feeding behavior. The ventral subregion of the hippocampus, vHP, is learned food intake. one critical site mediating ghrelin's stimulatory effects on conditioned Consistent with this model, delivery of ghrelin to the vHP activates feeding behavior, and ghrelin signaling in this brain region engages orexin dpLHA neurons that produce orexin, a neuropeptide that stimulates ap- signaling originating in the lateral hypothalamus to stimulate appetite – petite and feeding [127 133], and lateral ventricle delivery of an orexin- and ingestion. Future studies will aim to comprehensively understand 1 receptor antagonist (using a dose with no effect on feeding alone) other neural targets and interactions between endocrine neuropeptide blocked the hyperphagic effects of vHP ghrelin delivery [111].Thus,we systems through which ghrelin acts as a learned signal to feed. hypothesize that orexin is a critical downstream target for ghrelin- mediated conditioned feeding effects. As is the case with ghrelin receptor Acknowledgements knockout mice, orexin receptor mutation also reduces food anticipatory activity [127]. Moreover, it appears as though orexin, like ghrelin, can be We would like to acknowledge the support from the National Insti- entrained in response to restricted meal schedules and meal anticipation tute of Health grants DK104897 (SEK) and SSIB for the New Investigator [134,135]. The VTA is one putative site through which orexin signaling in- Travel Award (TMH). We report no biomedical financial interests or po- fluences learned feeding behavior, as blockade of orexin receptors in the tential conflicts of interest. VTA attenuates the hyperphagic response following ventricular delivery of ghrelin [136]. In addition to being responsive to vHP ghrelin signaling, orexin-producing neurons in the LHA express GHSR and are activated by References direct LHA ghrelin delivery [137], thus raising that possibility that ghrelin [1] C.E. Cornell, J. Rodin, H. 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Fig. 1. A model for ventral hippocampus (vHP) ghrelin-mediated conditioned feeding behavior (adapted from [111]): A) humans and animals learn to anticipate feeding based on external or internal food cues (e.g. habitual circadian cues, food advertisements, and sight and/or smell of food). B) Ghrelin secreted from the stomach in anticipation of feeding crosses the blood- brain barrier and enters the central nervous system. C) Ghrelin activates ghrelin receptors expressed on vHP pyramidal neurons, which directly engages D) downstream activation of dpLHA orexin neurons and orexin secretion. E) Activation of this neural pathway and downstream orexin receptor signaling F) enhances learned feedingbehavior.

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Please cite this article as: T.M. Hsu, et al., Ghrelin: A link between memory and ingestive behavior, Physiol Behav (2016), http://dx.doi.org/ 10.1016/j.physbeh.2016.03.039