The neuroanatomical function of leptin in the hypothalamus

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The neuroanatomical function of leptin in the hypothalamus M.M.H. van Swieten a,b, R. Pandit a, R.A.H. Adan a, G. van der Plasse a,* a

Department of Translational Neuroscience, Division of Neuroscience, Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht, The Netherlands b Master’s Program Neuroscience and Cognition, Utrecht University, The Netherlands

A R T I C L E I N F O

A B S T R A C T

Article history: Received 29 December 2013 Received in revised form 9 May 2014 Accepted 28 May 2014 Available online xxx

The anorexigenic hormone leptin plays an important role in the control of food intake and feedingrelated behavior, for an important part through its action in the hypothalamus. The adipose-derived hormone modulates a complex network of several intercommunicating orexigenic and anorexigenic neuropeptides in the hypothalamus to reduce food intake and increase energy expenditure. In this review we present an updated overview of the functional role of leptin in respect to feeding and feedingrelated behavior per distinct hypothalamic nuclei. In addition to the arcuate nucleus, which is a major leptin sensitive hub, leptin-responsive neurons in other hypothalamic nuclei, including the, dorsomedial-, ventromedial- and paraventricular nucleus and the lateral hypothalamic area, are direct targets of leptin. However, leptin also modulates hypothalamic neurons in an indirect manner, such as via the melanocortin system. The dissection of the complexity of leptin’s action on the networks involved in energy balance is subject of recent and future studies. A full understanding of the role of hypothalamic leptin in the regulation of energy balance requires cell-specific manipulation using of conditional deletion and expression of leptin receptors. In addition, optogenetic and pharmacogenetic tools in combination with other pharmacological (such as the recent discovery of a leptin receptor antagonist) and neuronal tracing techniques to map the circuit, will be helpful to understand the role of leptin receptor expressing neurons. Better understanding of these circuits and the involvement of leptin could provide potential sites for therapeutic interventions in obesity and metabolic diseases characterized by dysregulation of energy balance. ß 2014 Elsevier B.V. All rights reserved.

Keywords: Leptin Ob-R Feeding Hypothalamus Neuropeptides

1. Introduction 1.1. Leptin Abbreviations: AgRP, agouti related protein; Alpha-MSH, a-melanocyte-stimulating hormone; Arc, arcuate nucleus; AVP, arginine-vasopressin; BAT, brown adipose tissue; BBB, blood–brain-barrier; BDNF, brain derived neurotrophic factor; BNST, bed nucleus of the stria terminalis; CART, cocaine and amphetamine regulated transcript; CRH, corticotropin releasing hormone; DMH, dorsomedial hypothalamus; Dyn, dynorphin; GABA, g-aminobutyric acid; Gal, galanin; GALP, galanin-like peptide; GFP, green fluorescent protein; HPA, hypothalamic–pituitary–adrenal; HPT, hypothalamic–pituitary–thyroid; iBAT, interscapalur brown adipose tissue; icv, intracerebroventricular; IML, intermediolateral; LH, lateral hypothalamus; LS, lateral septum; MCH, melanin-concentrating hormone; ME, median eminence; NAc, nucleus accumbens; NPY, neuropeptide Y; NTs, neurotensin; NTS, nucleus of the solitary tract; OR, orexin; OXT, oxytocin; POMC, pro-opiomelanocortin; PVN, paraventricular nucleus; rRPa, raphe pallidus; SF-1, steroidogenic factor 1; SNA, sympathetic nervous activity; SNS, sympathetic nervous system; SON, supraoptic nucleus; TRH, thyroid releasing hormone; TrkB, tyrosine receptor B; VMH, ventromedial hypothalamus; VTA, ventral tegmental area. * Corresponding author at: Department of Translational Neuroscience, Division of Neuroscience, Brain Center Rudolf Magnus, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands. Tel.: +31(0)88 75 6 88 10. E-mail address: [email protected] (G. van der Plasse).

The anorexic hormone leptin is the product of the ob gene which is localized on the mouse and human chromosomes 6 and 7q31.3, respectively (Friedman et al., 1991; Green et al., 1995). Leptin is primarily expressed in white adipose tissue (Zhang et al., 1994) and functions as an afferent signal in the regulation of body weight, food intake and energy expenditure (Halaas et al., 1995; Pelleymounter et al., 1995). Circulating leptin concentrations are broadly proportional to the total amount of body fat and BMI (body mass index) in both rodents and humans (Considine et al., 1996; Maffei et al., 1995). As such, circulating leptin and leptin mRNA levels are highly correlated to energy stores in adipose tissue (Considine et al., 1996; Frederich et al., 1995; Maffei et al., 1995). In addition, overfeeding leads to elevated leptin levels whereas fasting reduces leptin availability (Kolaczynski et al., 1996; Saladin et al., 1995). Another factor that determines the level of circulating leptin is the size of the adipocytes. Larger adipocytes contain more

http://dx.doi.org/10.1016/j.jchemneu.2014.05.004 0891-0618/ß 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: van Swieten, M.M.H., et al., The neuroanatomical function of leptin in the hypothalamus. J. Chem. Neuroanat. (2014), http://dx.doi.org/10.1016/j.jchemneu.2014.05.004

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leptin in comparison with smaller adipocytes in the same individual (Considine et al., 1996; Madej et al., 1995). An important factor in leptin-mediated signaling is the passage of leptin across the blood–brain barrier. This is through active transport as leptin is too large to cross the blood–brain-barrier (BBB) by simple diffusion. The relatively permeable BBB of the arcuate nucleus (Arc) and median eminence (ME) enables leptin to enter the mediobasal hypothalamus (Banks et al., 1996) where it acts on the leptin receptor. It has been suggested that leptin is transported across the BBB in a dose-dependent manner by a unidirectional saturable system (Banks et al., 1996; Burguera and Couce, 2001; Maness et al., 1998). The short form of the leptin receptor, which is abundantly found at the BBB, has been indicated to be involved in the transport of leptin across the BBB (Bjørbæk et al., 1998; Boado et al., 1998; Kastin et al., 1999). Interestingly, Prevot and colleagues recently established a new physiological concept in the regulation of energy homeostasis. These authors showed that the nutritional status of an individual modulates the permeability of the blood–hypothalamus barrier allowing metabolic hormones to directly access a subset of Arc neurons. The Arc lies adjacent to the ME, which contains a blood– cerebrospinal fluid barrier composed of tanycytes, specialized ependymal cells located in the floor of the third ventricle, which allows for passive and rapid transport of circulating leptin into the medial basal hypothalamus (Prevot et al., 2013). The close proximity of the arcuate to the BBB, makes the arcuate nucleus a specially sensitive site for leptin. An unresolved question is how far leptin penetrates to other hypothalamic nuclei that express leptin receptors and whether the leptin predominantly enters the brain at sites where the BBB is thin, like near the arcuate nucleus. 1.2. Leptin receptor binding The db gene encodes the leptin receptor (Ob-R), which belongs to the class I cytokine receptor family (Tartaglia et al., 1995). Several isoforms of the Ob-R are produced by alternative splicing and at least six isoforms designated Ob-Ra-f, have been identified so far. The short form receptors, i.e. Ob-Ra, Ob-Rc, Ob-Rd and Ob-Rf, have been implicated in the transport of leptin across the BBB, as they are mainly expressed in the choroid plexus, vascular endothelium and peripheral tissues (Bjørbæk et al., 1998; Elmquist et al., 1998b; Kastin et al., 1999). The Ob-Re is a soluble receptor that enhances leptin’s half-life (Abbott et al., 2000; Bittencourt and Elias, 1998; Huang et al., 2001; Lammert et al., 2001; Yang et al., 2004) and serves as an antagonist of the transport of leptin (Tu et al., 2007). The long isoform Ob-Rb contains intracellular motifs necessary for activating the JAK/STAT signal transduction pathway (Bjørbæk et al., 1997) and is therefore essential for the physiological action of leptin in the hypothalamus, where it is expressed at high levels (Elmquist et al., 1998b). As the long isoform is the only functional leptin receptor in the brain this review will focus on its distribution and function. Unliganded Ob-Rb exists as a pre-formed homodimer. Each receptor can bind one leptin molecule with high affinity (Fong et al., 1998; Tartaglia et al., 1995). Upon leptin binding, the conformation of the Ob-Rb dimer changes, enabling transphosphorylation and activation of intracellular Ob-Rb-associated JAK2 molecules (Couturier and Jockers, 2003; Devos, 1997; Tartaglia, 1997). Furthermore, the intracellular domain of the Ob-Rb contains a binding site for the transcription factor signal transducer activator of transcription (STAT) proteins, including STAT3 (Bates and Myers, 2003), and mediates the translocation of STAT3 into the nucleus where it promotes the transcription of several genes, including suppressor of cytokine signaling 3 (SOCS3) (Bjorbak et al., 2000; Dunn et al., 2005). Leptin receptor activity is

negatively regulated by its own activity (Dunn et al., 2005) as SOCS3 suppresses the activity at the level of Tyr 985 and Box1 motif (Endo et al., 1997; Mu¨nzberg and Myers, 2005; Shimizu et al., 2007; Starr et al., 1997). In addition, the non-transmembrane enzyme phosphatase protein 1B (PTP1B) inhibits the activity of ObRbs at the level of the Box1 motif (Zabolotny et al., 2002). Direct and indirect activation of the JAK/STAT pathway activates multiple signaling pathways involving the activation of kinase-induced phosphorylation of proteins, including JAK2/STAT3, phosphoinositide 3-kinase (PI3K), extracellular signal-regulated kinase (ERK) and insulin receptor substrate 1 (IRS1). Interestingly, leptin directly activates PI3K in POMC neurons while inducing an opposite effect in AgRP neurons (Xu et al., 2005). For a more detailed overview of the intracellular signaling pathways activated by leptin (see Fru¨hbeck, 2006; Myers, 2004). 1.3. Leptin receptor distribution The functional importance of the hypothalamus for leptin’s role in energy homeostasis stems from its high leptin-receptor density in both rodents and humans (Elmquist et al., 1998b; Haˆkansson et al., 1998; Schwartz et al., 1996), enabling leptin to modulate a neuronal activity across multiple brain areas and cell types. In particular, leptin receptors are abundantly expressed in hypothalamic nuclei involved in the regulation of energy homeostasis including the Arc, ventromedial nucleus, dorsomedial hypothalamus and the lateral hypothalamic area (Elmquist et al., 1998b; Haˆkansson et al., 1998; Ha˚kansson and Meister, 1998; Mercer et al., 1996; Schwartz et al., 1996). In addition, Ob-R mRNA is also expressed in various degrees in extra-hypothalamic regions, including the cerebellum, hippocampus, amygdala, brains stem and substantia nigra (Elmquist et al., 1998b; Figlewicz et al., 2003; Grill and Kaplan, 2002; Haˆkansson et al., 1998; Mercer et al., 1996). 1.4. Hypothalamus The hypothalamus serves as the primary site for the integration and regulation of a multitude of physiological processes, including thermoregulation, metabolism, and body weight, aspects of cardiovascular function, physiologic adaptation to stress, regulation of growth and reproduction (including sexual behavior). Hypothalamic nuclei receive information from peripheral tissue through nervous connections, the bloodstream and the cerebrospinal fluid and respond to inputs via the same routes. The role of the hypothalamus in the regulation of body weight and food intake was established by lesion studies, highlighting the involvement of the Arc, the ventromedial- (VMH), and dorsomedial hypothalamus (DMH), the paraventricular nucleus (PVN) and the lateral hypothalamus (LH). All of which are areas that express the leptin receptor (Elias et al., 2000; Haˆkansson et al., 1998). In the following sections we will describe several of the key hypothalamic nuclei and outline the role of leptin signaling in their function. Although it is clear that leptin serves a multitude of functions, we will focus in this review on its role in feeding behavior and energy homeostasis. For more elaborate overview of the neural circuits involved in regulating feeding behavior see (Schwartz and Zeltser, 2013). Although various leptin-receptor expressing cell-types are found across multiple brain areas, in this review we will describe known cell-type specific modulation by leptin per hypothalamic area. Where possible we will elaborate on the functional consequences of leptin signaling. A detailed neuroanatomical map of leptin-receptor distribution is presented in Fig. 1. Table 1 presents an overview of the neuropeptides involved in energy balance with respective receptor expression and projections.

Please cite this article in press as: van Swieten, M.M.H., et al., The neuroanatomical function of leptin in the hypothalamus. J. Chem. Neuroanat. (2014), http://dx.doi.org/10.1016/j.jchemneu.2014.05.004

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Fig. 1. Schematic overview of the functional role of leptin in the hypothalamus. Red/‘’: orexigenic peptides inhibited by leptin, green/‘+’: anorexigenic peptides stimulated by leptin, Blue/‘+/’: unknown. Extra-hypothalamic projections involved in energy balance are not depicted. a-MSH, alpha-melanocyte stimulating hormone; AgRP, agoutirelated peptide; Arc, arcuate nucleus; AVP, arginine-vasopressin; BDNF, brain-derived neurotrophic factor; CART, cocaine- and amphetamine regulated transcript; CRH, corticotropin-releasing hormone; DMH, dorsomedial hypothalamus; Dyn, dynorphin; GABA, g-aminobutyric acid; Gal, galanin; GALP, galanin-like peptide; LH, lateral hypothalamus; MCH, melanin-concentrating hormone; NPY, neuropeptide Y; NTs, neurotensin; OXT, oxytocin; POMC, proopiomelanocortin; PVN, paraventricular nucleus; SF-1, steroidogenic factor-1; TRH, thyroid-releasing hormone; VMH, ventromedial nucleus. For references see Table 1.

2. Arcuate nucleus The arcuate nucleus (Arc) is one of the major targets for circulating leptin as the relatively permeable BBB in the Arc enables circulating leptin to access the hypothalamus. Functionally, Arc neurons play a key role in mediating the actions of leptin to regulate energy balance, glucose homeostasis and insulin levels. In the Arc, leptin acts on several distinct neuronal populations that co-express the Ob-Rb and, in part, exert opposite actions on the regulation of feeding behavior (Fig. 1). One population coexpresses POMC and CART (an anorectic peptide which will be discussed in detail in Section 2.1), inhibits food intake and is localized ventromedially in the Arc (Vrang et al., 1999). Secondly, NPY- and AGRP expressing neurons increase food intake upon activation and are localized more medially in the Arc (Broberger et al., 1997). In addition, leptin acts on galanin- and GALP neurons that have been shown to mediate orexigenic- and anorexigenic effects respectively. Of note, Arc neurons are inherently heterogeneous with respect to the expression of neurotransmitters. For example, a subpopulation of POMC neurons co-expresses glutamate, whereas NPY expressing neurons also express AGRP and gamma-aminobutyric acid (GABA). Interestingly, non-AgRP GABAergic neurons play an important role in the disinhibition of POMC neurons (Vong et al., 2011). Importantly, the melanocortin system acts as a downstream mediator of leptin in the regulation of energy balance (Cone, 2005).

Although five MC-Rs are identified, only MC3-R and MC4-R are expressed in the brain, including the hypothalamic nuclei implicated in energy homeostasis (Kishi et al., 2003; Mountjoy et al., 1994; Roselli-Rehfuss et al., 1993). MC3/4R are activated by the POMC product alpha-MSH (Adan et al., 1994; Marsh et al., 1999) and inhibited by AgRP (Yang et al., 1999). In the following sections we will describe leptin-mediated modulation within the Arc, as well as indirect modulation via efferent and afferent connections within the hypothalamus. 2.1. NPY/AgRP and POMC/CART The most widely studied neuronal subpopulations involved in feeding behavior are NPY/AgRP and POMC/CART neurons. POMC/ CART neurons provide an important anorexigenic regulatory mechanism of feeding behavior. Leptin directly activates POMC/ CART neurons to enhance POMC- (Thornton et al., 1997) and CART mRNA expression (Kristensen et al., 1998). Moreover, fasting decreases POMC and CART expression (Kristensen et al., 1998; Lambert et al., 1998; Volkoff and Peter, 2000). In addition, foodintake and body weight are decreased by alpha-MSH via MC4R signaling (Abbott et al., 2000; Cone, 2006; Mcminn et al., 2000) and CART (Kristensen et al., 1998), whereas the regulation of adiposity occurs via MC3R and MC4R signaling (Butler, 2006). In contrast, activation of AgRP neurons induces a strong feeding response that is mediated via release of NPY, AgRP and the

Please cite this article in press as: van Swieten, M.M.H., et al., The neuroanatomical function of leptin in the hypothalamus. J. Chem. Neuroanat. (2014), http://dx.doi.org/10.1016/j.jchemneu.2014.05.004

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Table 1 Overview of neuropeptides involved in energy balance with respective receptor expression and projections. Neuropeptide

Receptor

Co-express

Projection

References

Arcuate nucleus POMC

Ob-Rb

CART

LH

Cheung et al. (1997), Haˆkansson et al. (1998), Vrang et al. (1999), Elias et al. (1999, 2000), Elmquist (2001) Fekete et al. (2000) Enriori et al. (2011)

TRH (PVN) Preganglionic neurons (IML-spinal cord)

CART GALP

OR1R/OR2R MC3R NPY Y1 + Y5 Ob-Rb Ob-Rb NPY Y1 OR1R

NPY

Ob-Rb

GAL

OR1R MC4R Ob-Rb

Alpha-MSH

AgRP

GABA

Dorsomedial hypothalamus CART Ob-Rb

Ob-Rb BDNF GAL

Willie et al. (2001), Funahashi et al. (2003) Bagnol et al. (1999) Broberger et al. (1997), Fetissov et al. (2004), Roseberry et al. (2004) Kristensen et al. (1998) Takenoya et al. (2002) Cunningham (2004) Takenoya et al. (2003) Takenoya et al. (2005) Kuramochi et al. (2006) Elmquist et al. (1998b) Legradi and Lechan (1998), Elmquist et al. (1999) Atasoy et al. (2012) Elias et al. (1999), Broberger et al. (1998b) Vong et al. (2011) Willie et al. (2001), Funahashi et al. (2003) Cowley et al. (2001), Mounien et al. (2005) Laque et al. (2013)

OR + MCH (LH) NPY (DMH) PVN TRH (PVN) OXT (PVN) OR + MCH (LH) POMC

PVN Raphe pallidus (rRPa) + brown adipose tissue (BAT) NPY MCH

Lee et al. (2013b) Broberger (1999), Vrang et al. (1999) Kernie et al. (2000), Xu et al. (2003) Laque et al. (2013)

Ob-Rb

Ventromedial nucleus SF-1 Ob-Rb BDNF MC4R NPY Y1 Paraventricular nucleus TRH Ob-Rb

CRH

OXT AVP BDNF

NPY Y1 NPY Y5 MC4R Ob-Rb MC4R NPY Y1 Ob-Rb NPY Y5 Ob-Rb NPY Y5 NPY Y1 MC4R

Lateral hypothalamic area Orexin NPY Y1 + Y5

Dhillon et al. (2006) Xu et al. (2003) Chee et al. (2010)

CART CART

CART

Nuclear of solitary tract (NTS)

CART

Galanin/dynorphin VTA VMH POMC (Arc) NPY (Arc) GALP (Arc)

MCH

CART/GABA NPY Y1

Neurotensin Gal

Ob-Rb Ob-Rb

Kristensen et al. (1998), Gautron et al. (2010), Zhang et al. (2011) Enriori et al. (2011), Gautron et al. (2010), Zhang et al. (2011)

Gal/CART CART

NAc/striatum VTA + OR (LH)

neurotransmitter GABA. Of these, NPY is one of the most potent orexigenic neuropeptides, as intracerebroventricular (icv) injections of NPY stimulate feeding (Billington et al., 1991; Stanley and Leibowitz, 1985) and reduce energy expenditure (Billington et al., 1991). Icv injections of AgRP also triggers feeding behavior but over a longer timescale with slower onset (Semjonous et al., 2009) and recent data have implicated GABA as a key player in the orexigenic

Haˆkansson et al. (1998), Elmquist et al. (1998b), Broberger (1999), Vrang et al. (1999), Elias et al. (2001) Broberger (1999), Fekete (2002) Fekete (2002) Ghamari-Langroudi et al. (2010) Haˆkansson et al. (1998) Lu et al. (2003) Dimitrov et al. (2007) Haˆkansson et al. (1998), Vrang et al. (1999), Perello and Raingo (2013) Campbell et al. (2001) Haˆkansson et al. (1998), Vrang et al. (1999) Campbell et al. (2001) Wang et al. (2007) Xu et al. (2003)

Ha˚kansson et al. (1999), Chou et al. (2001) Dube et al. (2000), Jain et al. (2000) Funahashi et al. (2000) Funahashi et al. (2000) Funahashi et al. (2000), Muroya et al. (2004), Ma et al. (2007) Funahashi et al. (2000), Muroya et al. (2004) Takenoya et al. (2003) Broberger (1999), Vrang et al. (1999), Elias et al. (2001) Chaffer and Morris (2002), Gao et al. (2003) Georgescu et al. (2005), Sears et al. (2010) Jureus et al. (2000), Leinninger et al. (2009, 2011), Laque et al. (2013) Laque et al. (2013)

effects of AGRP neurons. Importantly, 98% of the AgRP neurons coexpress NPY (Bagnol et al., 1999; Broberger et al., 1998b) and twenty-six percent of the NPY/AgRP-containing neurons express the Ob-Rb (Funahashi et al., 2003). Functionally, leptin directly inhibits these neurons (Morrison et al., 2005; Schwartz et al., 1996) and NPY and AgRP mRNA-expression is enhanced as result of leptin deficiency or fasting (Mizuno and Mobbs, 1999; Stephens et al.,

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1995) (see Fig. 1). Of note, GABA is not only co-expressed in AgRP neurons, but also from a subset of AgRP-negative, GABA-positive neurons in the Arc that express the leptin receptor (Vong et al., 2011). Interestingly, leptin differentially modulates the NPY/AgRP neurons and POMC/CART neurons. Most of the evidence reported using electrophysiology, shows that leptin hyperpolarizes- and decreases the firing rate of Arc NPY (Spanswick et al., 1997), by activating an ATP-sensitive potassium channel in a PI3K-dependent manner (Harvey et al., 1997; Spanswick et al., 1997). The consequent efflux of K+ ions from the cell reduces the membrane potential and this hyperpolarization leads to a reduction of action potential frequency. In contrast, leptin administration to brain slices directly depolarizes POMC neurons via nonspecific cation channels. In addition, leptin acts on NPY-projecting inhibitory collaterals to POMC neurons which consequently reduce the release of GABA and NPY from the terminals (Cowley et al., 2001). With respect to the mechanism of action through which changes in activity of AgRP/NPY and POMC/CART neurons affect feeding, the targets of these neurons and the peptides they release should be considered. Through the use of genetically modified mice and optogenetic tools, it was recently shown that the acute onset of feeding behavior following AgRP-neuron activation can be strongly attenuated through blockage of NPY- as well as GABAergic signaling in the PVN (Atasoy et al., 2012; Krashes et al., 2013). In contrast to the acute induction of feeding through NPY and GABA signaling, the effects of AgRP on feeding are slower and suggested to be mediated via the MC4 receptor (Krashes et al., 2013; Semjonous et al., 2009). In addition, within the Arc, AgRP neurons also project directly onto POMC neurons (Cowley et al., 2001). Activation of these neurons can stimulate long-term feeding via GABA-mediated suppression of POMC signaling (Atasoy et al., 2012) and the subsequent reduction of alpha-MSH release (Cone, 2005; Jobst et al., 2004). Moreover, Y1 and Y5 receptors are expressed on POMC neurons (Broberger et al., 1997; Fetissov et al., 2004; Roseberry et al., 2004), suggesting a modulatory role for NPY in the inhibition of these neurons. Surprisingly, POMC neurons also express the MC3R, which acts as an auto-inhibitory receptor on POMC neurons (Bagnol et al., 1999). In addition, both MC3R and MC4R mRNA are expressed in NPY neurons, suggesting that alphaMSH may directly suppress the NPY activity via MC signaling (Cowley et al., 2001; Mounien et al., 2005). Specific deletion of Ob-R in POMC and NPY/AgRP neurons individually or combined, increases body weight and adiposity. Young mice lacking the Ob-Rb in POMC and NPY/AgRP neurons exhibit hyperphagia, decreased energy expenditure and increased weight gain, which is partially attenuated by high levels of leptin, indicating that the absence of leptin action in NPY/AgRP and POMC neurons is partially compensated by other leptin-regulated pathways (van de Wall et al., 2008). Ob-Rb deficiency in POMC and AgRP neurons results in hyperinsulinemia (van de Wall et al., 2008) while rescuing Ob-Rb expression in Arc neurons of Ob-Rb deficient rodents partially normalizes glucose and insulin levels (Coppari et al., 2005), demonstrating the involvement of leptin in the regulation of glucose homeostasis and insulin levels. Of functional importance is that the activity of Arc POMC and NPY/AgRP neurons is also modulated via leptin-regulated projections originating in other hypothalamic nuclei. Lateral hypothalamic orexin axon terminals are in contact with Arc NPY- and POMC neurons and orexin stimulates NPY neurons via orexin-1 receptors while inhibiting POMC neurons predominantly via orexin-2 receptors (Muroya et al., 2004; Willie et al., 2001). In addition, POMC neurons receive strong excitatory inputs from leptin-responsive neurons in the medial VMH and the strength of these inputs is diminished during fasting (Sternson et al., 2005). In contrast, NPY neurons are not innervated by the VMH, but receive

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weak inhibitory inputs from within the Arc (Sternson et al., 2005). Although previous research suggested melanocortins, AgRP and NPY as main downstream actors of leptin signaling, opto- and pharmacogenetic studies targeting leptin-responsive arcuate nucleus neurons now suggest a more important role for neurotransmitters such as GABA. 2.2. Galanin In addition to AgRP/NPY- and POMC/CART neurons, galanin (GAL) neurons in the Arc express the leptin receptor. GAL is a 29amino acid peptide widely expressed in the hypothalamus, including the Arc, PVN, DMH and LH (Gundlach et al., 1990). Evidence for galanin’s role in the regulation of feeding behavior is inconsistent and difficult to compare, due to different experimental approaches and anatomical areas investigated. In particular, information about its function within the Arc is sparse. In general, galanin is mildly orexigenic when injected icv or directly injected into the VMH and LH (Schick et al., 1993), where it possibly facilitates NPY signaling. However, the most prominent increase in food intake is observed when galanin is injected into the PVN, suggesting this is the primary nucleus mediating the orexigenic action of galanin (Kyrkouli et al., 2006, 1990). Leptin administration reverses galanin-induced food intake (Sahu, 1998b) and decreases galanin gene expression in the hypothalamus (Sahu, 1998a), indicating that galanin-expressing neurons are a direct target of leptin. Interestingly Cheung et al. (2001) report very low concentrations of Ob-Rb mRNA in galanin-expressing neurons, which may explain the unresponsiveness to food deprivation and changes in leptin levels compared to other orexigenic peptides, such as NPY, AgRP and MCH. Importantly, the immunohistochemical detection of the Ob-Rb is difficult due to the lack of specific and sensitive antibodies. To overcome this problem, a recent study by Laque et al. (2013) used Gal-Green Fluorescent Protein (GFP) and Ob-Rb GFP promoter mice to visualize the expression of leptin-induced pSTAT3 and galanin and to determine co-localization. Laque et al. (2013) demonstrated that leptin induces galanin expression and stimulates neuronal activity in Ob-Rb-expressing neurons in the ventral DMH, LH and at low levels in the Arc, which is conflicting with the orexigenic action of galanin reported before. In addition, galanin Ob-Rb-expressing neurons have recently also been found to colocalize with anorexigenic neuropeptides, including neurotensin and CART in the LH (Laque et al., 2013), underscoring its involvement in the anorectic action of leptin. Moreover, at least three galanin subpopulations are found in the LH, namely galanin/ CART-Ob-Rb and galanin/neurotensin-Ob-Rb and other galaninOb-Rb neurons, which are distinct from orexin and MCHexpressing neurons in the LH. Collectively, leptin’s action on galanin and the mechanisms involved in feeding behavior remain poorly understood. Specific targeting of these galanin subpopulations by using cre-mouse lines and optogenetics in combination with a behavioral assay may be necessary to shed light on its function. 2.3. Galanin-like peptide (GALP) Another subpopulation homologues to galanin is the galaninlike peptide (GALP), which also binds to galanin receptors in vitro (Ohtaki, 1999). Within the Arc, immunoreactivity studies show that GALP cell bodies are located in the media-posterior section (Takatsu et al., 2001). Importantly, more than 85% of GALP neurons express the Ob-Rb some of which are also POMC-positive (Takatsu et al., 2001). In addition, the number of cells containing GALP mRNA is reduced during fasting, whereas leptin treatment increases the number of cells expressing GALP mRNA in 4-fold

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(Jureus et al., 2000), suggesting that GALP is an anorexigenic peptide regulated by leptin. However, Matsumoto et al. (2002) reported that icv administration of GALP increases food intake in a manner that is ten-fold more potent than galanin. Furthermore, GALP neurons are heterogeneous in their projections as they innervate orexin- and MCH neurons in the LHA, neurons in the PVN (Takenoya et al., 2005) and NPY neurons in the DMH (Kuramochi et al., 2006). In addition, GALP neurons receive inputs from orexin neurons in the LH (Takenoya et al., 2003) and Arc NPY neurons (Cunningham, 2004). Moreover, double label in situ hybridization demonstrated that GALP-positive neurons express Y1 receptors (Cunningham, 2004) and orexin-1 receptors (Takenoya et al., 2003), their physiological actions remain uncertain, however. Taken together, through its action on GALP neurons, leptin may regulate both orexigenic (DMH NPY and/or orexin) and anorexigenic signals (alpha-MSH). 2.4. Projections to other hypothalamic areas The Arc is also essential in mediating the homeostatic responses of neurons through its extensive projections to other hypothalamic nuclei (see Fig. 1). Arc NPY/AgRP and POMC/CART neurons project to the PVN, LH, DMH and VMH (Bagnol et al., 1999; Elmquist et al., 1999). About 20% of the Arc NPY neurons innervate the PVN and DMH (Bai et al., 1985; Baker and Herkenham, 1995). Stimulation of this pathway enhances food intake through direct stimulation of Y1 and Y5 receptors in addition to AgRP antagonism of MC3R and MC4R in the PVN (Kalra and Kalra, 2003). Importantly, as discussed earlier, GABA was recently identified as a key neurotransmitter in the AgRP-PVN circuitry, projecting in particular oxytocin neurons, underlying acute AgRP neuron evoked eating (Atasoy et al., 2012). Both NPY/AgRP and POMC/CART neurons project to the PVN, in particular to corticotropin-releasing hormone (CRH) and thyroidreleasing hormone (TRH) in the PVN (Fekete et al., 2000; Legradi and Lechan, 1998; Le´gra´di and Lechan, 1999), which regulate the hypothalamic–pituitary–adrenal and hypothalamic–pituitary– thyroid axis, respectively through direct projections to the median eminence via the tuberoinfundibular system (Antoni et al., 1983; Toni and Lechan, 1993). This indicates that leptin in the Arc indirectly modulates the activity of these axes. Furthermore, projections from NPY/AgRP and POMC/CART in the Arc to orexin and MCH neuron in the LH have been identified (Broberger et al., 1998b; Elias et al., 1998b). As mentioned before, leptin directly and differentially engages NPY and POMC neurons that project to the LH, linking circulating leptin with neurons that regulate feeding behavior and body weight homeostasis (Elias et al., 1999). In addition, both Arc NPY and Arc POMC innervate the VMH. In particular, BDNF-producing neurons (discussed in more detail in Section 4.2) are regulated by NPY and alpha-MSH, through NPY Y1 (Chee et al., 2010) and MC4R (Xu et al., 2003) signaling, respectively. 3. Dorsomedial hypothalamus The DMH is involved in neuroendocrine and autonomic homeostasis. It is located caudal to the PVN and dorsal to the VMH. It has extensive intra-and extra-hypothalamic connectivity (Thompson et al., 1996) and is closely involved in various functions, including body weight regulation, ingestive behavior, thermoregulation, circadian rhythms, cardiovascular function, locomotor activity, stress and reproduction. Lesion studies have established the role of the DMH in the regulation of feeding and metabolism. In particular, animals exhibit hypophagia and reduced body weight after lesioning the DMH (Bellinger and Bernardis, 2002; Bellinger et al., 1986; Bernardis and Bellinger, 1998). Although it is reported that leptin

directly activates neurons in the DMH, the neurochemical phenotype of these leptin responsive neurons remains unclear (Elias et al., 1998b; Laque et al., 2013; Lee et al., 2013b; Patterson et al., 2011). In addition, a recent study using Ob-Rb-GFP mice showed that the Ob-Rb expressing neurons are located to the dorsal and ventral division in the caudal region of the DMH (Scott et al., 2009). In the DMH leptin acts as an important regulator of body weight and thermogenesis. In mice, icv injection of leptin decreases appetite and increases sympathetic nerve activity (SNA), leading to thermogenesis and increased energy expenditure in interscapular brown adipose tissue (iBAT) (Haynes et al., 1999; Zhang et al., 2011). Leptin’s involvement in heat production is thought to be direct via activation of Ob-Rb-expressing neurons in the DMH and indirectly via stimulation of alpha-MSH release by Ob-Rbexpressing Arc POMC neurons which subsequently activate DMH-, PVN- (see Fig. 1) and pre-ganglionic intermediolateral (IML) neurons that express MC4Rs. Both direct and indirect pathways are likely to mediate peripheral sympathetic outflow, resulting in increased expression and activity of mitochondrial uncoupling protein 1 (UCP-1) in brown adipose tissue (BAT) to produce heat (Enriori et al., 2011). Interestingly, in obese mice, selective leptin resistance occurs only in Arc neurons (AgRP and POMC neurons) (Mu¨nzberg et al., 2004). Other areas, including the DMH, are still responsive to leptin and therefore leptin-expressing neurons are able to increase sympathetic nervous outflow to iBAT during chronic hyperleptinemia found in obesity (Enriori et al., 2011). In addition, Ob-Rb-expressing neurons in the DMH project directly to neurons in the PVN and possibly premotor sympathetic neurons to increase SNA (Gautron et al., 2010). Furthermore, the raphe pallidus (rRPa), a region that contains premotor sympathetic neurons and innervates IML neurons in the spinal cord, is also innervated by Ob-Rb DMH neurons and are stimulated by cold exposure (Zhang et al., 2011). Although various cell-types have been indentified within the DMH, some of which also express the leptin receptor, only CART/NPY neurons are known to receive direct modulation from within the hypothalamus (see Fig. 1). In the next paragraphs, DMH CART and NPY will be described in more detail. 3.1. CART Cocaine-and-amphetamine-regulated transcript is one of the most abundantly expressed peptides in the hypothalamus that is involved in appetite regulation in rats and humans (Charnay et al., 1999; Hurd and Fagergren, 2000). In addition to the CART expression in the Arc, where it colocalizes with POMC, it is also found in the DMH, VMH, PVN, supraoptic nucleus (SON) and perifornical lateral hypothalamus (Elias et al., 2001; Koylu et al., 1997) (Fig. 1). The CART peptide is identified as an anorectic peptide which reduces food intake and regulates the hypothalamic–pituitary– adrenal (HPA) axis by activating CRH neurons in the PVN when injected icv (Kristensen et al., 1998; Stanley et al., 2001). CART expression is directly regulated by leptin (Elias et al., 2001; Kristensen et al., 1998) and is not dependent on melanocortin signaling, as CART-induced hypophagia is not blocked by antagonizing MC3R or MC4R with central AgRP (Edwards et al., 2000). Leptin induces and fasting inhibits CART mRNA expression in the Arc and more moderately in the DMH and PVN (Elias et al., 1998a, 2000; Kristensen et al., 1998). There is conflicting evidence on the interaction of CART with the NPY system. Although some studies show that NPY-positive terminals are in apposition of CART immunoreactive cell bodies in the PVN, DMH, LH and Arc (Broberger, 1999; Lambert et al., 1998), Lee et al. (2013b) demonstrated that NPY and CART are co-localized in the DMH

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and DMH NPY neurons are stimulated by leptin. Interestingly, CART co-localizes with both anorexigenic and orexigenic neuropeptides in the hypothalamus (Vrang et al., 1999). In the DMH, almost all the immunoreactivity of CART peptides is co-localized with melanin-concentrating hormone (MCH) (Broberger, 1999; Elias et al., 2001), however co-localization of CART and NPY immunoreactivity also exists (Lee et al., 2013b). Furthermore, CART mRNA and CART peptides are co-localized with POMC in the Arc, with MCH in the LH, and with TRH, galanin, vasopressin, and oxytocin in the PVN (Elias et al., 2001; Vrang et al., 1999). Despite extensive research on CART neurons, leptin’s action on CART neurons and mechanisms that mediate CARTs role in appetite regulation remain to be elucidated. 3.2. NPY In addition the presence of NPY neurons in the Arc, in situ hybridization histochemistry has revealed that NPY-expressing neurons are also localized in the DMH (Bi et al., 2003; White et al., 1990). However, NPY neurons in the Arc and DMH seem to differ in function. In contrast with the essential NPY mRNA expression in the Arc, NPY mRNA in the DMH is only expressed during chronic hyperphagic conditions as obesity (Guan et al., 1998), lactation (Elias et al., 1998a) and postnatal development (Grove et al., 2001) and during increased physical activity (Kawaguchi et al., 2005). Although the regulation of NPY neurons in the DMH was thought to be independent of leptin activation, Lee et al. (2013a) recently showed that NPY neurons co-localized with CART contain Ob-Rbs and are directly activated by leptin during chronic hyperphagic conditions. Interestingly, this study suggests that the expression of orexigenic NPY is driven by an unknown signal during the progression of diet induced obesity and that chronic hyperleptinemia increases the activity of NPY/CART neurons in the DMH. Importantly, NPY/CART neurons project to the PVN during chronic hyperphagic conditions (Lee et al., 2013a) and are implicated in body weight regulation by affecting food intake, thermogenesis and energy expenditure (Chao et al., 2011). Furthermore, DMH NPY neurons receive input from GALP neurons in the Arc (Shiba et al., 2010), indicating that leptin also regulates DMH NPY neurons indirectly. In addition, the DMH contains a high level of NPY- and alpha-MSH terminals projecting from the Arc (Bai et al., 1985; Gehlert et al., 1987; Jacobowitz and O’Donohue, 1978), which underscores the indirect actions of leptin. Moreover, these alpha-MSH fibers also project from the DMH to the PVN, terminating on the TRH-containing neurons (Miha´ly et al., 2001). 3.3. Projections to other hypothalamic nuclei Lesion studies have demonstrated that the DMH extensively projects to the PVN, in particular to regions involved in the control of the autonomic nervous system (Aravich and Sclafani, 1983; Bernardis and Bellinger, 1998). More specifically, a major projection pathway exists from leptin-responsive neurons in the DMH, to TRH-producing neurons in the PVN (Miha´ly et al., 2001), which suggests that the DMH is anatomically situated to exert a regulatory effect on TRH-synthesizing neurons in the PVN. In addition, leptin-responsive neurons in the DMH also project to the Arc and the PVN while smaller projections are found in the LH (DeFalco et al., 2001; Gautron et al., 2010). 4. Ventromedial hypothalamus The ventromedial hypothalamus (VMH) controls a number of homeostatic and autonomic behavioral responses, including the

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regulation of body weight and energy homeostasis. VMH lesions in rats stimulate appetite and cause obesity, while feeding and body weight is reduced after electrical stimulation. However, the VMH’s involvement in feeding behavior appears to be very complex as it contains a heterogeneous neuronal population, differing in their functional and biochemical identity. Although as many as nine subpopulations of neurons in the VMH have been identified based on their electrophysiological properties, it remains to be elucidated if these populations are functionally and neurochemically distinct (Sabatier and Leng, 2008). Although at least a portion of VMH/DMH neurons in vivo show to respond to both leptin and food availability (van der Plasse et al., 2013), their functional relation to feeding-related behavior is not completely understood. Functionally, at least a portion of these neurons have been shown to increase VMH neurons express different neuropeptides, receptors and transcription factors, including brain-derived neurotrophic factor (BDNF) and the transcription factor stereogenic factor-1 (SF1) which are involved in energy balance. Additionally, the regulation of food intake and body weight seems to be regulated in part through modulation of the autonomic nervous system, underlining the complex role of the VMH in feeding-related behavior (King, 2006). Although little is known about leptin action in the VMH, the identification of Ob-Rb in the VMH confirms its involvement in energy balance (Elmquist et al., 1998a). Of the entire population of VMH neurons, 25–30% of the glucoseresponsive and non-glucose-responsive neurons also express Ob-Rb mRNA (Kang et al., 2004). Furthermore, many leptinresponsive neurons are also sensitive to glucose (Kang et al., 2004; Shiraishi et al., 1999), indicating that the VMH is an important target site for the regulation of glucose homeostasis. Leptin administered into the VMH stimulates glucose uptake in peripheral tissue, including brown adipose tissue, heart and skeletal muscle (Toda et al., 2009). VMH leptin does so, at least in part, via modulation of sympathetic tone, as the leptin-induced increase in glucose uptake by BAT is suppressed in a denervated sympathetic nervous system (Minokoshi et al., 1999). Ob-Rbs are highly expressed in the dorsomedial region of the VMH (Elmquist et al., 1997, 1998b) and are directly activated by leptin as c-fos immunoreactivity and SOCS3 mRNA is detected following leptin administration (Elias et al., 2000; Elmquist et al., 1997; Hubschle et al., 2001). However, very little is known with respect to leptin’s function in the VMH. 4.1. SF1 neurons SF1 neurons in the VMH are first-order leptin-responsive neurons that mediate important anti-obesity effects of leptin as Ob-Rb deletion in SF1 neurons or SF1-deficient mice result in an obese phenotype (Bingham et al., 2008; Dhillon et al., 2006; Majdic et al., 2002). In addition, leptin increases the firing rate of SF1 positive neurons and activates the JAK/STAT signaling cascade, which subsequently activates several downstream proteins involved in the modulation of SF1 activity (Dhillon et al., 2006). Furthermore, mice lacking SOCS3 in SF-1 neurons show enhanced sensitivity to exogenous leptin and improved glucose homeostasis in the absence of body weight changes in both normal- and high fat chow-fed mice (Zhang et al., 2008), suggesting that SOCS3 in SF1 neurons negatively regulates leptin signaling and play an important role in mediating leptin sensitivity, glucose homeostasis and energy expenditure. In addition, mice lacking PI3K in VMH SF1 neurons show blunted responses to leptin and have disrupted regulation of energy expenditure. In particular, reduced PI3K activity in VMH neurons lead to a reduction in thermogenic functions of BAT by suppressing UCP1 expression (Xu et al., 2010). Taken together, PI3K activity in VMH neurons plays a physiological relevant role in

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the regulation of energy expenditure and mediates acute effects of leptin. 4.2. BDNF In addition to SF1 neurons, brain-derived neurotrophic factor (BDNF) containing neurons, are abundantly found in the VMH, as well as in the DMH and PVN (Kernie et al., 2000; Xu et al., 2003). BDNF is a neurotrophin that is widely expressed in the CNS and is crucial for neuronal development and plasticity (Barde, 1994) Although BDNF-producing neurons do not express Ob-Rbs, they are at least in part modulated by leptin as icv leptin injection increases BDNF mRNA expression and protein in the VMH (Komori et al., 2006). Leptin enhances BDNF production in an indirect manner by stimulating the release of alpha-MSH from the Arc, which acts on MC4R expressed by BDNF-producing neurons (Wisse and Schwartz, 2003; Xu et al., 2003), demonstrating its involvement in the regulation of energy balance as downstream target of leptin. Once BDNF is secreted, it interacts with its tyrosine receptor B (TrkB) located in the PVN, DMH, Arc and VMH, generating feedback stimulation to reduce food intake. In contrast, Arc NPY, which is negatively regulated by leptin, binds to NPY Y1 receptors expressed in the VMH to promote an orexigenic response by inhibiting the production of BDNF (Chee et al., 2010). This NPYBDNF interaction is supported by data from Wang et al. (2007) which demonstrates that BDNF infusion into the VMH suppresses NPY-induced feeding and body weight gain via TrkB signaling in combination with increased energy expenditure. In addition, BDNF expression is also regulated by nutritional state (Xu et al., 2003) and deficiency in BDNF expression causes obesity (Rios et al., 2001). 4.3. Projections to other hypothalamic nuclei Ob-Rb-expressing neurons in the medial VMH send strong excitatory innervations to POMC neurons, but not NPY neurons in the Arc (Sternson et al., 2005). Thus, leptin activates Arc POMC neurons directly as well as indirectly via the VMH. Furthermore, leptin-responsive neurons in the dorsomedial part of the VMH innervate the subparaventicular zone, providing a pathway by which leptin possibly regulate the secretion of hormones across the circadian cycle (Elmquist et al., 1998a). 5. Paraventricular nucleus of the hypothalamus The paraventricular nucleus of the hypothalamus (PVN) plays a major role in the control of both appetite and endocrine function. It contains distinct neuronal subpopulations that together account for the regulation of numerous physiological processes. The neurons in the PVN are one of the most characterized subcircuits involved in energy homeostasis. Magnocellular neurons in the PVN project to the posterior pituitary, where they release oxytocin and arginine vasopressin into the blood via hypophyseal capillaries. Parvocellular neurons in the PVN, including CRH and TRH-producing neurons, project to the median eminence, where they release hormones in the hypophyseal portal system to regulate hormone release from all endocrine cell types in the anterior pituitary. While the action of several pituitary hormones influence food intake indirectly via effects on energy expenditure, hormones of the HPA-axis modulate feeding behaviors directly. In vitro slice preparations revealed that the majority of neurons in the PVN are directly depolarized by leptin (Powis et al., 1998), indicating that they are targets of leptin signaling. In addition, most of these populations express MC-Rs and NPY receptor subtypes and receive dense projections from Arc POMC/CART and Arc NPY/AgRP neurons. As such, knockdown of

MC4R in the PVN promotes hyperphagia and obesity in response to the dietary challenge in the adult animal (Garza et al., 2008). In addition, the PVN is the main site of integration for visceral inputs from the sympathetic nervous system and nucleus of the solitary tract (NTS), cortico-limbic inputs from the lateral septum (LS) and bed nucleus of the stria terminalis (BNST) and interoceptive inputs from the Arc. 5.1. Thyroid-releasing hormone Thyroid releasing hormone (TRH) acutely stimulates the release of thyroid stimulating hormone (TSH) and prolactin from the anterior pituitary gland (Gershengorn, 1986). Within the PVN, leptin directly activates TRH neurons (Huo et al., 2004) and TRH inhibits food intake downstream of the leptin-melanocortin pathway (Harris et al., 2001). During fasting, thyroid function is decreased to reduce energy expenditure in humans and mice as low leptin levels are associated with a reduction of TRH gene expression and TRH formation (Harris et al., 2001; Huo et al., 2004; Le´gra´di et al., 1997; Perello et al., 2006). Indirect modulation of TRH neurons by leptin is mediated in a large part by projections from the Arc. Alpha-MSH released from these projection neurons induces neuronal activity in TRHexpressing neurons and TRH release which can be reversed by MC3R and MC4R antagonists (Kim et al., 2000). Interestingly, THR release is stimulated by alpha-MSH predominantly via MC4R, whereas AgRP inhibits TRH release mainly by acting on MC3R (Kim et al., 2002). Arc NPY input (Elias et al., 1999) promotes positive energy balance by suppressing TRH neurons in the PVN and HPT activity predominantly via NPY Y1 and Y5 receptors (Fekete, 2002) (Fig. 1). Moreover, NPY attenuates the alpha-MSH-induced increase in TRH production by decreasing the amount of alphaMSH and alpha-MSH-induced CREB phosphorylation in the PVN (Cyr et al., 2013). TRH neurons stimulate BDNF production in the PVN, where BDNF is abundantly expressed along with its receptor tyrosine receptor B (TrkB; Wang et al., 2007). Whereas these neurons are inhibited by NPY via NPY Y1 receptor signaling they are activated by alpha-MSH through melanocortin receptors (Wang et al., 2007; Xu et al., 2003). 5.2. Neurophysin neurons: oxytocin and arginine-vasopressin Oxytocin (OXT) and arginine-vasopressin (AVP) are expressed in both magnocellular and parvocellular neurons in the PVN. OXT is a nine amino acid peptide which is best known for its role in parturition and lactation. However, it also acts to reduce food intake (Arletti et al., 1989), an effect that can be blocked by icv injection of an oxytocin receptor antagonist (Olson et al., 1991). Leptin directly activates Ob-R expressed by magnocellular oxytocin neurons in the PVN (Haˆkansson et al., 1998) as STAT3 phosphorylation occurs in oxytocin neurons, and thereby inhibits food intake. Interestingly, only the subpopulation of oxytocin neurons that innervate the NTS are activated by leptin, suggesting that leptin’s role in regulating body weight is, at least in part, mediated by oxytocin neurons (Perello and Raingo, 2013). Moreover, oxytocin signaling appears to be important for leptin’s action on regulation of body weight in fasted rats (Perello and Raingo, 2013). AVP is a nine amino acid antidiuretic hormone homologues to OXT. AVP and CRH are co-localized in some parvocellular neurons in the PVN (Morimoto et al., 2000) and secreted into the hypophysial-portal circulation (Schwartz and Zeltser, 2013). Icv leptin administration enhances plasma AVP levels and AVP mRNA expression in the PVN (Yamamoto et al., 1999) and stimulates the HPA-axis indirectly through activation of AVP receptors located on

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CRH neurons in the PVN (Morimoto et al., 2000). Furthermore, inmmunoreactivity evidence suggests that AVP neurons coexpress Ob-Rb (Haˆkansson et al., 1998), demonstrating that AVP may be a direct target of leptin. Both magnocellular OXT and AVP neurons express NPY Y5 receptor immunoreactivity and NPY immunoreactive fibers are juxtaposed to magnocellular neurons expressing Y5-immunoreactivity, suggesting that NPY may directly stimulate OXT and AVP release through activation of Y5 receptors (Campbell et al., 2001). 5.3. Projections to other hypothalamic nuclei Although leptin-responsive neurons in the PVN are innervated by many neurons from distinct hypothalamic nuclei, not many efferent projections within the hypothalamus are found. The PVN serves as an important integrator for many autonomic processes and therefore mainly projects to extra-hypothalamic areas. 6. Lateral hypothalamus The lateral hypothalamus (LH) is a modulator of the autonomic and somatomotor systems, where it regulates skeletal and cardiovascular muscle, the adrenal gland and brown adipose tissue (Cerri and Morrison, 2005; Kerman et al., 2006; Kerman and Bernard, 2007; Oldfield et al., 2002). The LH innervates several hypothalamic nuclei, several regions in the cortex, the midbrain and the brainstem (Saper et al., 1979). Importantly, the LH also serves an important role in the regulation of energy balance, and, in fact, is historically referred to as the ‘feeding center’. Whereas damage to the LH suppresses feeding and consequently reduces body weight (Anand and Brobeck, 1951), stimulation of the LH causes a drive to eat. Expression of Ob-Rb’s in the LH is high (Elmquist et al., 1998b), and recently several neuronal populations that are (in)directly activated by leptin have been identified (e.g. Orexin-, neurotensin-, galanin- and CART neurons; see Fig. 1). Interestingly, selective knockdown of leptin receptors in the LH induces increased food intake and body weight in rats maintained on a high-fat diet, suggesting that the overconsumption of high-calorie food can be prevented by endogenous leptin signaling (Davis et al., 2011). 6.1. Neurotensin The majority of Ob-Rb-expressing neurons in the LH contain neurotensin, a 13 amino acids containing anorexigenic neuropeptide that is released during feeding (Leinninger et al., 2011) and is directly activated by leptin (Leinninger et al., 2011; Sahu, 1998a). Interestingly, similar to a general LH knockdown of Ob-Rb (Davis et al., 2011), mice with selective knockout of leptin receptors on neurotensin neurons exhibit increased food intake and body weight (Leinninger et al., 2011), indicating that the activation of these, GABAergic, neurotensin Ob-Rb neurons regulate energy balance via food intake and energy expenditure. In particular, an Ob-Rb expressing neurotensin subpopulation innervates the VTA to regulate the mesolimbic dopamine system and the motivation for food rewards (Leinninger et al., 2009, 2011). Moreover, this subpopulation also innervates orexin neurons in the LH, indicating that the activity of local orexin neurons and the expression of orexin are controlled by leptin action via the neurotensin Ob-Rb neurons (Leinninger et al., 2011). 6.2. Orexin Orexin-A is a 33-amino acid peptide of 3.2 kDa and is completely conserved among several mammalian species, including the rat, mouse and human. Orexin-B is a 28-amino acid peptide

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which is identical in the mouse and rat, but has two amino-acid substitutions in the human. Orexin-producing neurons are exclusively found in the perifornical area and the posterior and lateral hypothalamic area in rats and humans (Date et al., 1999; Elias et al., 1998b; Nambu et al., 1999; Peyron et al., 1998). Functionally, orexins are involved in energy homeostasis as icv injection of orexin induces feeding and leptin administration reduces preproorexin mRNA expression (Sakurai et al., 1998). While Sakurai et al. (1998) reported up-regulation of preproorexin mRNA upon fasting, Tritos et al. (2001) found no alteration in preproorexin expression, suggesting that preproorexin response to fasting may be in contrast to other orexigenic neuropeptides, such as NPY and AgRP, which are up-regulated upon fasting. Collectively, the modulation of orexin neurons by leptin appears to be complex. Early studies suggested that orexin neurons are colocalized with Ob-Rb (Haˆkansson et al., 1998), indicating that leptin directly inhibits the expression and/or secretion of orexins resulting in a reduction in food intake. However, recent data demonstrates that orexin neurons are distinct from Ob-Rbexpressing neurons in the LH (Leinninger et al., 2009), indicating that orexin neurons are indirectly modulated by leptin. In particular, a major neurotensin Ob-Rb-expressing subpopulation in the LH is implicated as the main neural mediator of orexin gene expression (Leinninger et al., 2011; Louis et al., 2010). Furthermore, orexin expression of normal and leptin-deficient mice is negatively correlated with changes in food intake and leptin levels (Yamanaka et al., 2003). More evidence for a functional relationship between leptin and orexin is obtained by studies in vitro and in vivo as leptin administration suppresses neuronal firing of isolated orexin neurons and fasting-induced activation of orexin neurons in vivo is blocked (Tritos et al., 2001; Yamanaka et al., 2003). Surprisingly, orexin signaling via orexin receptor 2 (OX2R) promotes negative energy balance and contributes to leptin anorectic actions (Funato et al., 2009). Furthermore, Ob-Rb expressing orexin neurons are co-localized with galanin (Ha˚kansson et al., 1999) and dynorphin (Chou et al., 2001) and are in apposition of MCH-containing neurons suggesting a possible interaction between orexin and MCH (Broberger et al., 1998a). In addition to the local innervations of LH neurotensin neurons, antero- and retrograde tracer studies have reported that orexin neurons are also innervated by a number of other neuronal populations (Sakurai et al., 2005; Yoshida et al., 2006) associated with the regulation of energy homeostasis, such as AgRP, NPY and alpha-MSH, presumably originated in the Arc (Broberger et al., 1998b; Elias et al., 1998b). 6.3. Melanin concentrating hormone (MCH) MCH is an orexigenic cyclic neuropeptide of 19 amino acids which acts as an anabolic signal in energy regulation, as it increases food intake in rodents when injected icv. MCH neurons are primarily located in the LH and zona incerta (Bittencourt and Elias, 1998). Two subpopulation of MCH neurons have been identified; one containing glutamate which projects to the cortex and another population which co-expresses CART and contains GABA and projects to the brainstem (Cvetkovic et al., 2004; Elias et al., 2001). MCH neurons in the LH are orexigenic and regulated by leptin. Leptin suppresses MCH-induced food intake (Sahu, 1998b), reduces MCH gene expression (Sahu, 1998a) and normalizes the elevated levels of MCH and MCH receptor 1 (MCHR1) in leptin deficient mice (Huang et al., 1999; Kokkotou et al., 2001; Tritos et al., 2001). However, recent evidence indicates that MCH neurons do not contain the functional Ob-Rb (Leinninger et al., 2009), suggesting that the modulation of MCH neurons by leptin is indirect. Moreover, MCH neurons are indirectly regulated by

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inputs from Arc POMC and NPY/AgRP neurons (Broberger et al., 1998a; Elias et al., 1998b). 6.4. Projections to other hypothalamic nuclei Neurons in the LH project to other hypothalamic nuclei, in particular the Arc where MCH and orexin neurons innervate POMC and NPY/AgRP neurons (Broberger et al., 1998a; Elias et al., 1998b). In addition, whole cell patch clamp recordings demonstrate that orexin neurons suppress spontaneous firing in Arc POMC neurons in mouse brain slices, indicating that the orexigenic effects of orexin in part is established by suppressing neuronal activity of hypothalamic POMC neurons (Ma et al., 2007). 7. Concluding remarks In conclusion, through this review we outline how one peripherally circulating factor can differentially regulate various aspects of energy balance: from food intake to energy expenditure and from motivation to thermogenesis. Leptin is a prime example of how the body fine-tunes metabolism depending on the needs of the organism by selectively modulating various neuronal projections. The review also goes on to demonstrate the complexity of the physiology of body weight regulation and thus calls for future studies directed toward understanding the role of these individual pathways. In this review we presented an overview of the role of hypothalamic leptin signaling in relation to feeding and energy homeostasis. Leptin acts in the hypothalamus onto both orexigenic and anorexigenic networks of intercommunicating neurons, to control feeding and energy homeostasis. In particular, leptin suppresses the activity of orexigenic-, while stimulating the activity of the anorexigenic network, to establish a reduction in food intake and an increase in energy expenditure. In addition, the connectivity of leptin-responsive neurons with the autonomic and mesolimbic systems indicates that leptin action in the hypothalamus regulates a range of physiological processes. Importantly, the current review limits its focus to leptin’s action within the hypothalamus. The network controlling food intake and body weight is more complex and extends beyond the hypothalamus. In particular, through modulation of the autonomic nervous system (e.g. Ghamari-Langroudi et al., 2011; Leinninger and Myers, 2008) and midbrain dopamine system (e.g. DiLeone et al., 2003; Harris et al., 2005; van Zessen et al., 2012) leptin can act to control feeding behavior and energy expenditure. Although progress has been made in understanding the functional role of leptin signaling in feeding behavior, the functional consequences of cell-type specific leptin receptor binding are still poorly understood. For an important part this is due to the fact that leptin acts on populations of neurons that show great diversity in their neurochemical makeup and downstream targets. However, with the advent of pharmaco- and optogenetic tools great progress is currently made in targeting specific celltypes and projections to unravel the function of these leptinmodulated networks. In this respect, the use of neuropeptides as markers for specific cell-types and the promotors driving neuropeptide expression are particularly useful. Through the use of cre-mouse lines, specific populations of neurons can be targeted and brought under opto- or pharmacogenetic control. LepR-cre mice, for instance, are important to target different leptin receptor expressing neurons by site-specific injections of viral vectors conditionally expressing DREADDs or light-sensitive channels. Similarly, the use of leptin receptor antagonists or viral downregulation, will further drive our understanding of leptin’s functions.

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Please cite this article in press as: van Swieten, M.M.H., et al., The neuroanatomical function of leptin in the hypothalamus. J. Chem. Neuroanat. (2014), http://dx.doi.org/10.1016/j.jchemneu.2014.05.004