br Introduction Calorie restriction CR in the absence of mal

Introduction Calorie restriction (CR), in the absence of malnutrition, has beneficial effects on Phenformin function, including reducing the incidence of age-related neurodegenerative disease (Gräff et al., 2013), eliciting anti-depressant behavior (Lutter et al., 2008) and improving memory function in rodents (Fontán-Lozano et al., 2007). In non-human primates, prolonged CR in adulthood decreases the incidence of age-related disease, including measures of brain atrophy (Colman et al., 2009). Whilst in adult humans a 3-month period of CR has been shown to improve memory function (Witte et al., 2009). The physiological mechanism(s) underlying these effects are not fully understood. One process implicated in regulating anxiolytic and mnemonic behavior is adult hippocampal neurogenesis (AHN). This is a form of ongoing plasticity that occurs throughout life involving the birth, differentiation and maturation of new neurons in the adult mammalian dentate gyrus (DG). Decreased neurogenesis has been implicated in the pathogenesis of anxiety and depression (Snyder et al., 2011) as well as cognitive impairment (Yassa et al., 2011) and dementia (Höglinger et al., 2004, Komuro et al., 2015). Recently it has been shown that AHN is essential for distinguishing similar but distinct contexts by laying down non-overlapping memory traces (Clelland et al., 2009, Creer et al., 2010, Nakashiba et al., 2012, Sahay et al., 2011); this form of cognition, termed pattern separation, is impaired in anxiety-disorders (Kheirbek et al., 2012) and cognitive decline (Yassa et al., 2011). Notably, factors such as exercise (Van Praag et al., 2005, Van Praag et al., 1999) and environmental enrichment positively modulate the rate of AHN and performance in pattern separation dependent cognitive tasks (Clelland et al., 2009) and anxiety related tests (Llorens-martín et al., 2010). In addition, reducing the number of calories consumed promotes the survival of newborn cells in the hippocampus (Lee et al., 2002). However, the underlying mediator(s) are not known and it is unclear whether these newborn cells mature into differentiated neurons or if they integrate into hippocampal circuitry to modulate mnemonic processes. The orexigenic gastrointestinal hormone, acyl-ghrelin, which is elevated during CR (Lutter et al., 2008), is known to cross the BBB and bind to the growth hormone secretagogue receptor (Ghsr) within the hippocampus (Diano et al., 2006). Ghsr is necessary for the anxiolytic effect of CR and exogenous treatment with acyl-ghrelin reduces anxiety behavior (Lutter et al., 2008) and improves performance in spatial learning tasks (Carlini et al., 2010, Diano et al., 2006). Moreover, acyl-ghrelin increases cell proliferation in the hippocampus (Moon et al., 2009) and adult ghrelin deficient mice show reduced rates of new neuron differentiation that were restored to wild-type levels following acyl-ghrelin treatment (Li et al., 2013). These data demonstrate that supra-physiological doses of acyl-ghrelin improve cognition, however, more recently we showed that daily injections of acyl-ghrelin, at a dose similar to plasma concentrations after a 24h fast, enhanced AHN and pattern separation memory performance (Kent et al., 2015). Based on these data we hypothesize that acyl-ghrelin mediates the neurogenic and cognitive enhancing effects of CR. Here, we demonstrate that Ghsr is expressed in mature granule cells of the DG and that elevating peripheral acyl-ghrelin, either by injection or CR, increases expression of the zinc finger transcription factor, early growth response 1 (Egr-1) in the DG. Egr-1 is an immediate early gene involved in mitogenesis and differentiation that has recently been implicated in increasing AHN in mice (Veyrac et al., 2013). Using a two-week CR paradigm, paired with a BrdU pulse-chase approach, we demonstrate that CR increases the subsequent generation of adult born mature neurons in a Ghsr-dependent manner. Furthermore, the increase in hippocampal plasticity was accompanied by enhanced remote contextual fear memory, a mnemonic process associated with AHN (Kitamura et al., 2009). Together, these results show that Ghsr is required to mediate the beneficial effects of CR on hippocampal plasticity and memory.