Integration of metabolic stimuli in the hypothalamic arcuate nucleus

Kalsbeek, Fliers, Hofman, Swaab, Van Someren & Buijs Progress in Brain Research, Vol. 153 ISSN 0079-6123 Copyright r 2006 Elsevier B.V. All rights reserved

CHAPTER 8

Integration of metabolic stimuli in the hypothalamic arcuate nucleus M. van den Top and D. Spanswick Division of Clinical Sciences, Warwick Medical School, The University of Warwick, Coventry CV4 7AL, UK

Abstract: Integration of peripheral and central anabolic and catabolic inputs within the hypothalamic arcuate nucleus (ARC) is believed to be central to the maintenance of energy balance. In order to perform this complex task, neurons in the ARC express receptors for all major humoral and central transmitters involved in the maintenance of energy homeostasis. The integration of these inputs occurs at the cellular and circuit level and the resulting electrical output forms the origins for the activation of feeding and energy balance-related networks. Here, we discuss the role that active intrinsic membrane conductances, KATP channels and intracellular second messenger systems play in the integration of metabolic stimuli at the cellular level in the ARC. We conclude that the research into the integration of hunger and satiety signals in the ARC has made substantial progress in the last decade, but we are far from unraveling the complex neuronal networks involved in the maintenance of energy homeostasis. The diverse range of inputs, neuronal integrative properties, targets, output signals and how these signals relate to the physiological output provides us with a colossal challenge for years to come. However, to battle the current obesity epidemic, target-specific drugs need to be developed for which the knowledge of neuronal pathways involved in the maintenance of energy homeostasis will be crucial. Keywords: arcuate nucleus; active membrane conductances; ATP-sensitive potassium channels; leptin; glucose; PI3K Energy homeostasis is maintained by balancing food intake with energy expenditure resulting in consistent levels of stored energy in the form of fat. In order to maintain this balance the central nervous system (CNS) needs to integrate information originating from the periphery that signals energy status at the level of the central metabolic drive. Thus, a complex feedback system has evolved that utilizes humoral and neuronal signaling to inform the CNS of the whole body metabolic status. Hormones such as insulin, leptin, ghrelin and Peptide YY3 36 (PYY3 36) are secreted from the periphery in quantities related to metabolic status and vagal

pathways relay information from the gut back to the CNS, through the brainstem (for review see Broberger, 2005). Subsequently, neurons and their associated circuits within function-specific areas of the autonomic subdivision of the CNS sense, process and formulate anabolic or catabolic outputs. These outputs, as part of a complex neuronal network, drive the appropriate changes relative to the energy status of the organism and as such maintain energy homeostasis. Aspects of the CNS therefore function as key integrative centers responsible for information processing and the coordination of appropriate output. The arcuate nucleus (ARC) of the hypothalamus is such an integrative center.

Corresponding author. Tel.: +44 2476 572538; Fax: +44 2476 523701; E-mail: [email protected] DOI: 10.1016/S0079-6123(06)53008-0

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The best described anabolic neurons residing in the ARC use Neuropeptide Y and agouti-related protein (NPY/AgRP) as neurotransmitters/neuromodulators (Broberger et al., 1998; Morton and Schwartz, 2001). Proopiomelanocortin (POMC)expressing neurons, which can potentially cleave a range of neurotransmitters, are by far the best described catabolic neurons (Cone, 2005). Together, these two populations of neurons form parallel and opposing pathways that work together to maintain energy balance. However, a number of different neuronal networks originating within the ARC are involved in the maintenance of energy balance (Howard et al., 2000; Gottsch et al., 2004), although an in-depth understanding of the functioning of these networks is presently lacking. Within the ARC, the complexity of the neuronal networks controlling energy balance is reflected by the inputs, outputs and integrative properties of individual ARC neurons. At the cellular level, the physiological properties of ARC neurons and cellular mechanisms by which they integrate the multitude of orexigenic and anorexigenic signals remains poorly understood. A number of studies have shown that gene-expression characteristics of ARC neurons change in response to altered energy status or hormones, such as leptin and insulin (Schwartz et al., 1997; Kristensen et al., 1998; Mizuno et al., 1998; Mizuno and Mobbs, 1999; Howard et al., 2000; Jureus et al., 2000; Shimokawa et al., 2003). The changes in the expression levels of neuronal neuropeptidergic transmitters under various metabolic regimens have been shown to be a reliable tool to predict the overt function of these neurons. However, how these changes in metabolic status translate into appropriate modulation of electrical output and neurotransmitter release onto target neurons is unclear. The electrical activity generated in ARC neurons depends on the neurons’ intrinsic electrical properties, the type of input, the spatial organization and functional relationship between these properties and the relationship of the individual neuron with other component neurons forming the function-specific circuits. Taken together these neuronal properties are crucial for a neuron and its associated circuit to function as an integrative center for metabolic stimuli. The complexity of

this integrative process and the resulting output signal is reflected in the diversity of neuronal components controlling this process and the extent of the input and output pathways involved. A detailed review of the efferent and afferent pathways associated with the ARC is outside the remit of this chapter (for comprehensive reviews of this subject, see Chronwall, 1985; Broberger and Hokfelt, 2001) and thus we will highlight only a few facets of these pathways to illustrate the complexity of the integration processes performed by ARC neurons at the single cell level. To illustrate the complexity of the inputs to the ARC we will touch on the inputs originating from lateral hypothalamus (LH) and circulating factors. Such inputs utilize a combination of transmitters including fast neurotransmitters (gamma aminobutyric acid (GABA) and glutamate), neuropeptides (including orexin, melanin-concentrating hormone (MCH), Dynorphin-A, cocaine- and amphetamine-regulated transcript (CART) and in the case of circulating factors, hormones such as leptin, insulin, PYY3 36 and ghrelin (Elias et al., 1998; Peyron et al., 1998; Broberger, 1999; Date et al., 1999; Horvath et al., 1999; Chou et al., 2001; Elias et al., 2001; Hewson et al., 2002; Rosin et al., 2003). The general circulation contains a range of messengers that modulate the central metabolic drive through an action on neurons in the CNS including leptin, insulin, ghrelin and glucose (for review, see Woods et al., 1996; Elmquist et al., 1998; Horvath et al., 2001; Levin et al., 2004). The ARC is ideally situated and equipped to sense these humoral factors as it is located in a part of the brain with an impaired blood–brain barrier and neurons in the ARC express some of the highest levels in the CNS of receptors for circulating factors, including the anorexigens leptin (Ob-Rb) and insulin, and the orexigen ghrelin (growth hormone secretagogue receptor) (van Houten et al., 1980; Mercer et al., 1996; Willesen et al., 1999; Shuto et al., 2002). The humoral factors are all released in quantities relative to the nutritional and peripheral metabolic status, whereas in the CNS neurotransmitters are differentially released following the integration of the peripheral signals within the central drive. The inputs to the ARC of central origin comprise a multitude of transmitters and originate from a

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range of sources as outlined above. These transmitters are commonly colocalized, for example orexin colocalizes with glutamate and dynorphin-A, while CART colocalizes with MCH (Broberger, 1999; Chou et al., 2001; Rosin et al., 2003). The LH is classically referred to as a feeding center, which is highlighted by the fact that the majority of neuropeptides expressed in this hypothalamic nucleus are orexigenic. However, CART is an anorexigenic neuropeptide which is coexpressed with the orexigenic neuropeptide MCH (Qu et al., 1996; Kristensen et al., 1998; Shimada et al., 1998; Broberger, 1999). The functional significance of this coexpression of peptides with apparently opposing functions is presently unclear. The output of ARC neurons is distributed to functionally related areas in the CNS including: other hypothalamic nuclei such as the LH, paraventricular nucleus (PVN) and dorsomedial nucleus (DMN) with which the ARC reciprocally interacts; the brainstem and spinal cord (Bai et al., 1985; Baker and Herkenham, 1995; Beck, 2001). In addition to the NPY/AgRP and POMCderived peptides, other energy balance-related neurons originating here utilize galanin-like peptide (GALP), neuromedin U and CART that colocalizes with POMC. (Chronwall, 1985; Baker and Herkenham, 1995; Broberger et al., 1998; Legradi and Lechan, 1999; DeFalco et al., 2001) These neuropeptides are coexpressed with fast neurotransmitters GABA and glutamate as well as other neuropeptides, thus adding to the complexity, diversity and computational capability of these neurons and their associated circuits (Horvath et al., 1997; Elias et al., 1998; Hahn et al., 1998; Cowley et al., 2001; Collin et al., 2003; Hentges et al., 2004). How the differential release of neurotransmitters in the CNS relates to the dynamics of the neuronal activity of the neurons expressing them is poorly understood. For example, in neurosecretory neurons the release of neuropeptides has been shown to be dependent on the pattern of neuronal activity rather than the rate of activity (Dutton and Dyball, 1979; Bicknell and Leng, 1981; Poulain and Wakerley, 1982). In order to understand the role ARC neurons play in coordinating the central response to maintain energy balance, a greater appreciation of the functional organization of these neurons and their circuits, the dynamics of their output and

input and the cellular signal transduction mechanisms by which these neurons integrate metabolic signals is required. In this chapter, we will focus on the integrative properties of ARC neurons at a membrane level and signal transduction level.

Electrophysiological properties of ARC neurons The function, integration and computational capability of a neuron depends on its architecture and passive and active membrane properties, all of which contribute to its ability to instigate, modulate and set limits of sensitivity to detection of external stimuli. We have studied the electrophysiological and morphological properties of large number of ARC neurons maintained in rat hypothalamic slice preparations in vitro. ARC neurons have cell body sizes ranging from 10 to 30 mm with one to five fine neuronal projections. These neurons express a diversity of membrane resistances or input conductances ranging from 420 to 4667 MO suggesting heterogeneity within the population. Similarly, ARC neurons express a range of active membrane conductances of which several are readily identifiable in the current–voltage relationships (Fig. 1) These active conductances are differentially expressed, again indicating functional heterogeneity. In the absence of active membrane conductances negative current injections induced membrane potential responses directly proportional to the amount of current injected. However, the activation of certain active conductances results in nonlinear relationships between current and voltage and/or modulates the return to resting membrane potential following hyperpolarization. Differentially expressed active conductances observed in ARC neurons include anomalous inward rectification (IAN), hyperpolarization-activated non-selective cation conductances (IH), transient outward rectification (IA) and low voltage-activated T-type calcium conductances (IT) (Fig. 1). In some ARC neurons, the decrease in input resistance at more negative membrane potentials was instantaneous and non-inactivating and indicated by a decrease in the slope of the plot of the current–voltage relationship (Fig. 1Ai). The conductance resembles those described in other

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Fig. 1. Active conductances in ARC neurons. Ai: Current–voltage (I–V) relationship of an ARC neuron expressing anomalous inward rectifying and A-like potassium conductances. The plot shows superimposed traces from a continuous current clamp recording in which negative rectangular-wave current injections of constant increment induced a non-linear decrease in input resistance at more negative membrane potentials as a result of the activation of IAN. Arrow (1) indicates the decrease in the peak amplitude of membrane response at negative holding potentials (downward arrow) relative to the responses seen near resting membrane potential (upward arrow). Arrow (2) indicates the delayed return to baseline following negative current injection as a result of the activation of an A-like conductance. Aii: Plot of the I–V relationship obtained from the neuron shown in Ai. The reduced slope of the plot at more negative holding potentials is the result of the activation of IAN. Bi: I–V relationship of an ARC neuron expressing IH and IT. Note the sag in the membrane response during negative current injection as a result of the activation of IH. Rebound depolarizations were induced following the release of the holding current due to the activation of an IT and the slow inactivation of IH (Arrow (1)). Bii: Plot of the current–voltage relationship of the neuron shown in Bi.

neurons of the CNS including hypothalamic nuclei following activation of the so-called ‘‘anomalous inward rectifier,’’ which comprises potassium channels that effectively allow passage of potassium ions against its concentration gradient (Constanti and Galvan, 1983; Tasker and Dudek, 1991). The function of IAN is unclear, but as it acts to prevent cells becoming ‘‘too’’ hyperpolarized,

this conductance has been suggested to maintain the membrane potential of neurons in a functional range through its activation at negative membrane potentials. At the break of negative current injection a subpopulation of ARC neurons show a rebound excitation. The properties of the rebound depolarization are similar to those described resulting from the activation of IT (Llinas and Yarom, 1981; Llinas and Jahnsen, 1982). IT are implicated in the generation of burst-like firing patterns in neurons of the CNS (Huguenard, 1996). Moreover, IT can modulate the properties of synaptic inputs in the form of both inhibitory post-synaptic potentials (IPSPs) and excitatory post-synaptic potentials (EPSPs) (Huguenard and Prince, 1994; Gillessen and Alzheimer, 1997). Another conductance differentially expressed in ARC neurons is a time- and voltage-dependent inward rectification. The characteristics of the inward rectification resemble the features of IH (Halliwell and Adams, 1982; Pape, 1996). IH is a non-selective cation conductance involved in the generation of pacemaker activity, as described in the thalamus (Pape, 1996), and for its ability to modulate synaptic inputs (Magee, 1998, 1999). Transient outward rectifying conductances are a further feature of a subpopulation of ARC neurons. The conductance, observed at the break of the membrane response to negative current injection in ARC neurons as a delayed return to rest of the membrane potential, resembles the activation of A-like potassium conductances (Fig. 1; Connor and Stevens, 1971; Rogawski, 1985). Indeed, A-like conductances have been identified in a number of other hypothalamic nuclei and mouse ARC neurons (Bourque, 1988; Bouskila and Dudek, 1995; Luther et al., 2000; Burdakov et al., 2003). IA channels are classically involved in the repolarizing phase of the action potential and are thus important in the regulation of repetitive firing (Rogawski, 1985). Mouse and most rat Arc neurons express an A-like conductance similar to those classically reported for these currents (Burdakov et al., 2003 and our unpublished observations). The type, properties and location of active conductances expressed together with the location, size and kinetics of the synaptic input determine

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the final integrative properties of ARC neurons. In this respect the subset of ARC NPY/AgRPexpressing neurons in the ARC that contain an orexigen-sensitive pacemaker which we recently identified are of interest (van den Top et al., 2004). These neurons generated a burst-firing pattern in the presence of the orexigens, orexin and ghrelin (Fig. 2) and are inhibited by the catabolic hormone leptin (Fig. 5A). Within these neurons the active

Fig. 2. Pacemaker activity in the ARC. A: Trace showing typical burst firing as observed in orexigen-sensitive NPY/AgRP pacemaker neuron (top trace). Underlying oscillations in membrane potential were revealed by injection of negative current (bottom traces). B: Continuous current clamp recording showing burst firing induced in ARC pacemaker neurons in the presence of ghrelin. Subpopulations of ghrelin-responsive ARC neurons respond differentially in the presence of this orexigenic neuropeptide. The bottom trace shows an ARC neuron that is depolarized by ghrelin resulting in action potential firing in a repetitive manner.

conductances IA and IT play a key role in the generation and properties of the membrane potential oscillations underlying burst firing. Thus, blocking IT prevents the generation of membrane potential oscillations, while blocking IA increases the frequency of these events. The IA underlying the pacing of membrane potential oscillations has unusual properties, compared to other ARC neurons, in that these conductances can be activated around and more negative than rest and show an unusually long inactivation. At present it is unclear what underlies these differential properties of IA in pacemaker neurons compared to similar conductances in other neurons activated at more depolarized membrane potentials. Functionally, these neurons may form the origin of the central drive to feed although it remains to be established where these neurons project to and what transmitters are released through the generated burst firing. However, both orexin and ghrelin are believed to mediate their orexigenic effects through a central mechanism including the direct activation of NPY/AgRP neurons and subsequent downstream activation of NPY receptors (Dube et al., 2000; Jain et al., 2000; Yamanaka et al., 2000). Moreover, orexin neurons impinge directly onto ARC NPY neurons and ARC neurons, including pacemaker neurons, express orexin and ghrelin receptors (Horvath et al., 1999; Marcus et al., 2001; Chen et al., 2004; van den Top et al., 2004). Therefore, conditional pacemaker activity generated by NPY/AgRP neurons in response to orexigens may underpin the release of NPY and AgRP. Furthermore, the ability to modulate this activity through external metabolic and neurotransmitter signals provides a substrate to differentially regulate release thus adding to the level of functional plasticity and computational power of the system.

Modulation of synaptic inputs by active conductances ARC neurons receive inputs from both GABAergic and glutamatergic neurons observed as IPSPs and EPSPs, respectively the origins of which remain to be determined (Fig. 3; Kiss et al., 2005).

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Fig. 3. Chemical synaptic inputs to ARC neurons. Ai: Continuous current clamp recording showing spontaneous inhibitory postsynaptic potentials (IPSPs, downward deflection) and the subsequent block of these events in the presence of bicuculline methiodide (BMI), a blocker of GABAA receptors. A section of the trace is shown under the trace on an expanded time scale. Aii: Continuous current clamp recording in which the summation of IPSPs results in the induction of a rebound excitation and the discharge of an action potential. Bi: Trace from a current clamp recording showing spontaneous excitatory post-synaptic potentials (EPSPs, upward deflections) and the subsequent block of these events in the presence of NBQX, a blocker of non-NMDA glutamate receptors. The downward deflections are membrane responses to repetitive negative rectangular wave current injections.

EPSPs are blocked by the application of 6-nitro-7sulphamoylbenzo(f)-quinoxaline-2,3-dione (NBQX) suggesting them to be resulting from the activation of AMPA-type glutamate receptors (Fig. 3B). The IPSPs can be blocked with bicuculline methiodide (BMI), a GABAA receptor blocker, and IPSPs are generally assumed to reduce neuronal activity (Fig. 3Ai). However, functional significance of such inputs can only be realized when the intrinsic integrative properties of neurons are taken into account (Huguenard, 1996; Pape, 1996; Magee, 1998; van den Top et al., 2004). For example, as reported in other tissues, single or summated IPSPs can trigger rebound excitations in ARC and other neurons (Fig. 3Aii, Whyment et al., 2004). These rebound excitations have been reported to be mediated through the activation of IH and/or IT following the IPSP-induced transient membrane hyperpolarization (Huguenard and Prince, 1994). Recently, leptin was shown to rapidly modulate the synaptic inputs to ARC neurons in that leptin increases the number of inhibitory synapses onto

NPY/AgRP neurons, while reducing the excitatory inputs to POMC neurons (Pinto et al., 2004). However, to what extent this apparent synaptic plasticity plays in information processing in the ARC or its overall contribution to information processing is difficult to reconcile without understanding the degree of ‘‘hard-wiring’’ in the ARC pathways and the contribution of more conventional means of synaptic plasticity, such as preand post-synaptic modulation of inputs. Thus, precisely how neurons retract synaptic inputs and then rewire them in a functionally relevant manner to the post-synaptic cell to allow for integration by intrinsic conductances within the cell needs to be explored further. Indeed, in ghrelin excited and thus presumed orexigenic neurons in the ARC of Zucker fa/fa rats, which lack a functional long form of the Ob-Rb leptin receptor, we observe ghrelin-induced IPSPs and EPSPs similar to the ones observed in Zucker lean rats (Fig. 4; Phillips et al., 1996). These findings imply that the reported fast rewiring of the ARC does not occur in all neuronal populations and circuits of the ARC. As

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Fig. 4. Synaptic hardwiring in the ARC. Left: Continuous current clamp recordings showing induction of an excitation in conjunction with an increase in IPSPs in Zucker obese (fa/fa) and lean rats, respectively. The traces shown on an expanded time base and scale were taken at the peak of the response. Right: Trace showing the induction of EPSPs in Zucker lean and obese rats, respectively. Traces shown on an expanded time and voltage base are taken prior to (Top) and in the presence of ghrelin (Bottom). These findings suggest that the synaptic connections formed in the presence and absence of central leptin signaling are similar.

NPY and POMC neurons are not homogeneous neuronal populations, the discrepancy between our findings and those reported previously might partially lie in the fact that we have recorded from different neuronal subpopulations (Horvath et al., 1997; Collin et al., 2003; Hentges et al., 2004; our unpublished observations).

KATP channels and central integration of humoral factors So far, we have discussed the effects that membrane components of ARC neurons play in the

integration of inputs. However, intracellular signaling pathways are involved in this process too. The CNS needs to integrate peripheral inputs signaling the energy reserve status with central and peripheral short-term satiety/hunger inputs to maintain energy homeostasis. Classic examples of humoral factors that feedback information concerning the energy status are insulin, leptin and glucose. Insulin and leptin receptors are highly expressed in the ARC making this nucleus a prime target for the integration of these humoral factors into the central metabolic drive and maintain energy balance. The signal transduction pathways activated by insulin and leptin include common components as

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observed in the periphery i.e. both involve the activation of phosphoinositide 3-kinase (PI3K) (Niswender and Schwartz, 2003). Thus, if leptin and insulin impinge on the same target they may be capable of modulating their respective functions as a result of the cross talk between their intracellular-signaling cascades. This could, for example explain central leptin and insulin resistance observed in the obese (Munzberg and Myers, 2005). In the hypothalamus, the cross talk between the leptin and insulin intracellular-signaling pathways was originally investigated utilizing electrophysiological-recording techniques (Spanswick et al., 2000). Previously, Spanswick et al. (1997) had reported the activation of ATP-sensitive potassium channels (KATP) by leptin in glucose responsive neurons (GR-neurons) located in the ARC and the ventromedial nucleus of the hypothalamus (VMN). Subsequently, insulin was found to inhibit GR-neurons through a PI3K-dependent activation of KATP channels. Moreover, these two circulating anorectic hormones were shown to impinge on the same ARC neurons in a mutually exclusive manner. Finally, in line with previous findings in which insulin was shown to be incapable of reducing food intake, insulin was found unable to activate KATP in Zucker fa/fa rats (Ikeda et al., 1986; Spanswick et al., 2000). Recently, the interest in both (i) the role of PI3K and (ii) the role that KATP channels play in the hypothalamic control of energy homeostasis has gained momentum. (i) Studies investigating the role of PI3K in the leptin- and insulin-induced anorectic response that this enzyme is necessary for their anorectic response (Niswender et al., 2001, 2003). However, as discussed above the maintenance of energy balance involves subpopulations of neurons in the ARC of which neurons containing POMC (catabolic) and NPY/AgRP (anabolic) form parallel and apposing pathways that are differentially modulated by leptin. Thus, leptin/insulin inhibit the expression of NPY/AgRP, while they activate the expression of POMC in ARC neurons. Similarly, at the level of the cellular membrane leptin differentially modulates the neuronal activity of ARC POMC and NPY/AgRP neurons (Fig. 5A) (Cowley et al., 2001; van den Top et al., 2004). In

Fig. 5. Opposing effects of leptin on ARC neurons. A: (Top) Continuous current clamp recording showing the slow leptin-induced (50 nM) membrane potential hyperpolarization associated with a reduction in membrane resistance. In ARC pacemaker neurons, leptin induced a membrane potential hyperpolarization. Subsequent orexin application induced pacemaker activity. (Bottom) Continuous current clamp recording showing a leptin-induced (50 nM) excitation associated with a reduction in membrane resistance. B: Leptin excites ARC neurons through the activation of a non-selective cation channel. Superimposed plots of I–V relationships obtained in the presence and absence of leptin. Plot of these I– V relationships reveals an extrapolated reversal potential for the leptin-induced conductance of  25 mV.

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agreement with previous experiments performed on POMC neurons in mice, we also found in rat that the leptin-induced excitation is induced through the activation of a non-selective cation channel (Fig. 5B; Cowley et al., 2001). Both NPY/ AgRP and POMC neurons express leptin and insulin receptors thus raising the question, how identical receptors can impose opposite neuronal responses. Moreover, in both these neuronal populations leptin and insulin application results in a modulation of the PI3K activation level (Xu et al., 2005). Unfortunately, at present it is unclear how PI3K differentially modulates neuronal activity in NPY and POMC neurons. However, recently it has been shown that this enzyme mediates leptininduced inhibition of NPY and AgRP levels in the ARC. Logically, the neuronal activity will follow/ induce the changes in neuropeptide expression levels in order to achieve release, thus suggesting that the inhibition of NPY/AgRP neurons is likely to be mediated through an effect mediated via PI3K. (ii) Studies investigating the role the mediobasal hypothalamus in the control of hepatic glucose production have revealed a key role for KATP channels (Obici and Rossetti, 2003). Neurons sensing circulating levels of insulin and long-chain fatty acids are located in the mediobasal hypothalamus, which utilize KATP channels to modulate their neuronal activity in order to control hepatic glucose production (Obici et al., 2002a, b, c; Lam et al., 2005; Pocai et al., 2005). These findings, in combination with our previous results regarding the action of leptin and insulin in the ARC and VMN, imply KATP channels to be a common neuronal target for humoral factors in the CNS. However, although there are binding sites for fatty acids on KATP channels, it remains to be shown that this results in the modulation of these potassium channels in the ARC (Branstrom et al., 1997). Moreover, channels are widely expressed in the CNS including the hypothalamic ARC and do not form a functionally homogeneous population. In the ARC, electrophysiological studies have shown that diazoxide-sensitive KATP channels are expressed in the majority of neurons including neurons that express NPY/AgRP and POMC, respectively (Fig. 6; Ibrahim et al., 2003; Thomzig et al., 2005; our unpublished observations). Thus,

Fig. 6. The majority of ARC neurons express functional KATP channels. A: Continuous recording in current clamp (top three traces) showing the induction of a membrane hyperpolarization associated with a reduction in membrane resistance following a reduction of the extracellular glucose concentration from 10 to 0 mM (glucose free). The downward deflections are the membrane potential responses to repetitive negative rectangular wave current injections utilized to monitor input resistance. Note that the membrane response is gradually reduced as the membrane hyperpolarization progresses, indicating a reduction in membrane resistance. The application of the KATP channel blocker tolbutamide (100 mM) or the reintroduction of 10 mM glucose reverses the glucose free-induced response. This response was observed in 70% of the ARC neurons tested and included orexigen-sensitive NPY/AgRP pacemaker neurons. B: Current clamp recording performed in the presence of the sodium channel blocker tetrodotoxin (TTX) to electrically isolate the neuron, showing that diazoxide (500 mM) directly induced hyperpolarization of ARC neurons through the activation of KATP channels. Note the reduction in input resistance associated with this response. The application of tolbutamide (200 mM) reverses the effects of diazoxide.

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central diazoxide application as reported recently to elucidate the role of central KATP channels in the control of hepatic glucose production is likely to result in the inhibition of a number of functionally distinct neuronal populations in the ARC. In order to have a coordinated output from the ARC-opposing pathways like the NPY/AgRP and POMC, neurons need to be differentially modulated. However, how this is achieved remains to be clarified. In addition to representing a functional endpoint for leptin and insulin signaling, the KATP channel has classically been linked to glucose-sensing both in the periphery and in the CNS (Levin et al., 2004; Ashcroft, 2005). Centrally, the VMN has historically attracted the most attention but recent studies indicate a potential role for the ARC. The compromised blood–brain barrier here coupled with the widespread expression of KATP channels render this nucleus ideally situated and equipped to detect changes in circulating glucose levels. A recent study suggested that ARC neurons can detect physiological changes in glucose levels via a KATP-dependent mechanism (Wang et al., 2004). However, the inability to identify the functional phenotype of the neurons responding, given the widespread distribution of the channels, renders the observations difficult to interpret in a functional context. In order to elucidate the role of KATP channels expressed in ARC neurons in the maintenance of energy homeostasis, we need to establish how these channels are differentially activated under physiologically relevant conditions.

Future perspectives Since the discovery of leptin, the level of research activity and developments in our understanding of the central control of energy balance has expanded at an incredible rate. Unfortunately, so has our appreciation of the level of complexity we are dealing with. While our knowledge concerning the neurotransmitters and neurohormonal signals involved, their receptors and signal transduction mechanisms has improved considerably our understanding of the functional organization and operation of the neuronal components and their

circuits through which these systems operation has lagged behind. Electrophysiological studies in the ARC have described the effects of many feeding-related ligands on the electrical activity of ARC neurons including NPY, orexin, leptin, insulin, glucose, cholecystokinin and opioids (Kelly et al., 1990; Spanswick et al., 1997, 2000; Kinney et al., 1998; Cowley et al., 2001; Burdakov and Ashcroft, 2002; Burdakov et al., 2003; Ibrahim et al., 2003; Poulain et al., 2003; Roseberry et al., 2004; Wang et al., 2004). More recently, the development of transgenic mice that selectively express green fluorescent protein in POMC or NPY-containing neurons and single cell reverse transcriptase-polymerase chain reaction (RT-PCR) techniques has provided us with powerful tools to study functionally different neuronal populations (Cowley et al., 2001; Ibrahim et al., 2003; van den Top et al., 2004; Acuna-Goycolea et al., 2005; Takahashi and Cone, 2005). These latter advances will undoubtedly assist in the quest to understand the fundamental properties of ARC neurons and associated circuits controlling energy balance and the cellular- and molecular-signaling mechanisms by which these neurons coordinate a central response to perturbations in energy balance. However, significant investment is required to understand the operation of these neurons, their organization and operation in function-specific circuits and how these component parts detect, process, integrate and formulate appropriate output to perturbations in energy balance. The introduction of mathematical-modeling techniques and the development of predictive models will also greatly assist in the evolution of our understanding of the central control of energy balance and how disruption or dysfunction to this system manifests as obesity.

Abbreviations AgRP AMPA ARC ATP BMI

agouti-related peptide a-amino-3-hydroxy-5-methyl4-isoxazole proprionic acid arcuate nucleus adenosine triphosphate bicuculline methiodide

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CART CNS DMN EPSPs GABA GALP GR-neurons IA IAN IH IT IPSPs KATP LH MCH a-MSH NBQX NMU NPY Ob-Rb PI3K POMC PVN PYY3 36 RT-PCR TTX VMN

cocaine- and amphetamine-regulated transcript central nervous system dorsomedial nucleus of the hypothalamus excitatory post-synaptic potentials gamma aminobutyric acid galanin-like peptide glucose responsive neurons transient outward rectification anomalous inward rectification hyperpolarization-activated nonselective cation channel low voltage-activated T-type calcium conductance inhibitory post-synaptic potentials ATP-sensitive potassium channels lateral hypothalamus melanin concentrating hormone a-melanocyte stimulating hormone 6-nitro-7-sulphamoylbenzo(f)quinoxaline-2,3-dione neuromedin U neuropeptide Y long-form of the leptin receptor phosphoinositide 3-kinase proopiomelanocortin paraventricular nucleus of the hypothalamus peptide YY3 36 reverse transcriptase-polymerase chain reaction tetrodotoxin ventromedial nucleus of the hypothalamus

Acknowledgments The financial support from the Biotechnology and Biological Sciences Research Council (BBSRC), the Foundation for Prader-Willi Research and British Heart Foundation (BHF) is gratefully acknowledged.

References Acuna-Goycolea, C., Tamamaki, N., Yanagawa, Y., Obata, K. and van den Pol, A.N. (2005) Mechanisms of neuropeptide Y, peptide YY, and pancreatic polypeptide inhibition of identified green fluorescent protein-expressing GABA neurons in the hypothalamic neuroendocrine arcuate nucleus. J. Neurosci., 25: 7406–7419. Ashcroft, F.M. (2005) ATP-sensitive potassium channelopathies: focus on insulin secretion. J. Clin. Invest., 115: 2047–2058. Bai, F.L., Yamano, M., Shiotani, Y., Emson, P.C., Smith, A.D., Powell, J.F. and Tohyama, M. (1985) An arcuatoparaventricular and -dorsomedial hypothalamic neuropeptide Y-containing system which lacks noradrenaline in the rat. Brain Res., 331: 172–175. Baker, R.A. and Herkenham, M. (1995) Arcuate nucleus neurons that project to the hypothalamic paraventricular nucleus: neuropeptidergic identity and consequences of adrenalectomy on mRNA levels in the rat. J. Comp. Neurol., 358: 518–530. Beck, B. (2001) KOs and organisation of peptidergicfeeding behavior mechanisms. Neurosci. Biobehav. Rev., 25: 143–158. Bicknell, R.J. and Leng, G. (1981) Relative efficiency of neural firing patterns for vasopressin release in vitro. Neuroendocrinology, 33: 295–299. Bourque, C.W. (1988) Transient calcium-dependent potassium current in magnocellular neurosecretory cells of the rat supraoptic nucleus. J. Physiol., 397: 331–347. Bouskila, Y. and Dudek, F.E. (1995) A rapidly activating type of outward rectifier K+ current and A-current in rat suprachiasmatic nucleus neurones. J. Physiol., 488(Part 2): 339–350. Branstrom, R., Corkey, B.E., Berggren, P.O. and Larsson, O. (1997) Evidence for a unique long chain acyl-CoA esterbinding site on the ATP-regulated potassium channel in mouse pancreatic beta cells. J. Biol. Chem., 272: 17390–17394. Broberger, C. (1999) Hypothalamic cocaine- and amphetamineregulated transcript (CART) neurons: histochemical relationship to thyrotropin-releasing hormone, melanin-concentrating hormone, orexin/hypocretin and neuropeptide Y. Brain Res., 848: 101–113. Broberger, C. (2005) Brain regulation of food intake and appetite: molecules and networks. J. Intern. Med., 258: 301–327. Broberger, C. and Hokfelt, T. (2001) Hypothalamic and vagal neuropeptide circuitries regulating food intake. Physiol. Behav., 74: 669–682. Broberger, C., Johansen, J., Johansson, C., Schalling, M. and Hokfelt, T. (1998) The neuropeptide Y/agouti gene-related protein (AGRP) brain circuitry in normal, anorectic, and monosodium glutamate-treated mice. Proc. Natl. Acad. Sci. USA, 95: 15043–15048. Burdakov, D. and Ashcroft, F.M. (2002) Cholecystokinin tunes firing of an electrically distinct subset of arcuate nucleus

152 neurons by activating A-type potassium channels. J. Neurosci., 22: 6380–6387. Burdakov, D., Liss, B. and Ashcroft, F.M. (2003) Orexin excites GABAergic neurons of the arcuate nucleus by activating the sodium–calcium exchanger. J. Neurosci., 23: 4951–4957. Chen, H.Y., Trumbauer, M.E., Chen, A.S., Weingarth, D.T., Adams, J.R., Frazier, E.G., Shen, Z., Marsh, D.J., Feighner, S.D., Guan, X.M., Ye, Z., Nargund, R.P., Smith, R.G., Van der Ploeg, L.H., Howard, A.D., MacNeil, D.J. and Qian, S. (2004) Orexigenic action of peripheral ghrelin is mediated by neuropeptide Y and agouti-related protein. Endocrinology, 145: 2607–2612. Chou, T.C., Lee, C.E., Lu, J., Elmquist, J.K., Hara, J., Willie, J.T., Beuckmann, C.T., Chemelli, R.M., Sakurai, T., Yanagisawa, M., Saper, C.B. and Scammell, T.E. (2001) Orexin (hypocretin) neurons contain dynorphin. J. Neurosci., 21: RC168. Chronwall, B.M. (1985) Anatomy and physiology of the neuroendocrine arcuate nucleus. Peptides, 6(Suppl 2): 1–11. Collin, M., Backberg, M., Ovesjo, M.L., Fisone, G., Edwards, R.H., Fujiyama, F. and Meister, B. (2003) Plasma membrane and vesicular glutamate transporter mRNAs/proteins in hypothalamic neurons that regulate body weight. Eur. J. Neurosci., 18: 1265–1278. Cone, R.D. (2005) Anatomy and regulation of the central melanocortin system. Nat. Neurosci., 8: 571–578. Connor, J.A. and Stevens, C.F. (1971) Voltage clamp studies of a transient outward membrane current in gastropod neural somata. J. Physiol., 213: 21–30. Constanti, A. and Galvan, M. (1983) Fast inward-rectifying current accounts for anomalous rectification in olfactory cortex neurones. J. Physiol., 335: 153–178. Cowley, M.A., Smart, J.L., Rubinstein, M., Cerdan, M.G., Diano, S., Horvath, T.L., Cone, R.D. and Low, M.J. (2001) Leptin activates anorexigenic POMC neurons through a neural network in the arcuate nucleus. Nature, 411: 480–484. Date, Y., Ueta, Y., Yamashita, H., Yamaguchi, H., Matsukura, S., Kangawa, K., Sakurai, T., Yanagisawa, M. and Nakazato, M. (1999) Orexins, orexigenic hypothalamic peptides, interact with autonomic, neuroendocrine and neuroregulatory systems. Proc. Natl. Acad. Sci. USA, 96: 748–753. DeFalco, J., Tomishima, M., Liu, H., Zhao, C., Cai, X., Marth, J.D., Enquist, L. and Friedman, J.M. (2001) Virus-assisted mapping of neural inputs to a feeding center in the hypothalamus. Science, 291: 2608–2613. Dube, M.G., Horvath, T.L., Kalra, P.S. and Kalra, S.P. (2000) Evidence of NPY Y5 receptor involvement in food intake elicited by orexin A in sated rats. Peptides, 21: 1557–1560. Dutton, A. and Dyball, R.E. (1979) Phasic firing enhances vasopressin release from the rat neurohypophysis. J. Physiol., 290: 433–440. Elias, C.F., Lee, C., Kelly, J., Aschkenasi, C., Ahima, R.S., Couceyro, P.R., Kuhar, M.J., Saper, C.B. and Elmquist, J.K. (1998) Leptin activates hypothalamic CART neurons projecting to the spinal cord. Neuron, 21: 1375–1385. Elias, C.F., Lee, C.E., Kelly, J.F., Ahima, R.S., Kuhar, M., Saper, C.B. and Elmquist, J.K. (2001) Characterization of

CART neurons in the rat and human hypothalamus. J. Comp. Neurol., 432: 1–19. Elmquist, J.K., Maratos-Flier, E., Saper, C.B. and Flier, J.S. (1998) Unraveling the central nervous system pathways underlying responses to leptin. Nat. Neurosci., 1: 445–450. Gillessen, T. and Alzheimer, C. (1997) Amplification of EPSPs by low Ni(2+)- and amiloride-sensitive Ca2+ channels in apical dendrites of rat CA1 pyramidal neurons. J. Neurophysiol., 77: 1639–1643. Gottsch, M.L., Clifton, D.K. and Steiner, R.A. (2004) Galaninlike peptide as a link in the integration of metabolism and reproduction. Trends Endocrinol. Metab., 15: 215–221. Hahn, T.M., Breininger, J.F., Baskin, D.G. and Schwartz, M.W. (1998) Coexpression of Agrp and NPY in fasting-activated hypothalamic neurons. Nat. Neurosci., 1: 271–272. Halliwell, J.V. and Adams, P.R. (1982) Voltage-clamp analysis of muscarinic excitation in hippocampal neurons. Brain Res., 250: 71–92. Hentges, S.T., Nishiyama, M., Overstreet, L.S., Stenzel-Poore, M., Williams, J.T. and Low, M.J. (2004) GABA release from proopiomelanocortin neurons. J. Neurosci., 24: 1578–1583. Hewson, A.K., Tung, L.Y., Connell, D.W., Tookman, L. and Dickson, S.L. (2002) The rat arcuate nucleus integrates peripheral signals provided by leptin, insulin, and a ghrelin mimetic. Diabetes, 51: 3412–3419. Horvath, T.L., Bechmann, I., Naftolin, F., Kalra, S.P. and Leranth, C. (1997) Heterogeneity in the neuropeptide Y-containing neurons of the rat arcuate nucleus: GABAergic and non-GABAergic subpopulations. Brain Res., 756: 283–286. Horvath, T.L., Diano, S., Sotonyi, P., Heiman, M. and Tschop, M. (2001) Minireview: ghrelin and the regulation of energy balance — a hypothalamic perspective. Endocrinology, 142: 4163–4169. Horvath, T.L., Diano, S. and van den Pol, A.N. (1999) Synaptic interaction between hypocretin (orexin) and neuropeptide Y cells in the rodent and primate hypothalamus: a novel circuit implicated in metabolic and endocrine regulations. J. Neurosci., 19: 1072–1087. Howard, A.D., Wang, R., Pong, S.S., Mellin, T.N., Strack, A., Guan, X.M., Zeng, Z., Williams Jr., D.L., Feighner, S.D., Nunes, C.N., Murphy, B., Stair, J.N., Yu, H., Jiang, Q., Clements, M.K., Tan, C.P., McKee, K.K., Hreniuk, D.L., McDonald, T.P., Lynch, K.R., Evans, J.F., Austin, C.P., Caskey, C.T., Van der Ploeg, L.H. and Liu, Q. (2000) Identification of receptors for neuromedin U and its role in feeding. Nature, 406: 70–74. Huguenard, J.R. (1996) Low-threshold calcium currents in central nervous system neurons. Annu. Rev. Physiol., 58: 329–348. Huguenard, J.R. and Prince, D.A. (1994) Intrathalamic rhythmicity studied in vitro: nominal T-current modulation causes robust antioscillatory effects. J. Neurosci., 14: 5485–5502. Ibrahim, N., Bosch, M.A., Smart, J.L., Qiu, J., Rubinstein, M., Ronnekleiv, O.K., Low, M.J. and Kelly, M.J. (2003) Hypothalamic proopiomelanocortin neurons are glucose responsive and express K(ATP) channels. Endocrinology, 144: 1331–1340.

153 Ikeda, H., West, D.B., Pustek, J.J., Figlewicz, D.P., Greenwood, M.R., Porte Jr., D. and Woods, S.C. (1986) Intraventricular insulin reduces food intake and body weight of lean but not obese Zucker rats. Appetite, 7: 381–386. Jain, M.R., Horvath, T.L., Kalra, P.S. and Kalra, S.P. (2000) Evidence that NPY Y1 receptors are involved in stimulation of feeding by orexins (hypocretins) in sated rats. Regul. Peptides, 87: 19–24. Jureus, A., Cunningham, M.J., McClain, M.E., Clifton, D.K. and Steiner, R.A. (2000) Galanin-like peptide (GALP) is a target for regulation by leptin in the hypothalamus of the rat. Endocrinology, 141: 2703–2706. Kelly, M.J., Loose, M.D. and Ronnekleiv, O.K. (1990) Opioids hyperpolarize beta-endorphin neurons via mu-receptor activation of a potassium conductance. Neuroendocrinology, 52: 268–275. Kinney, G.A., Emmerson, P.J. and Miller, R.J. (1998) Galanin receptor-mediated inhibition of glutamate release in the arcuate nucleus of the hypothalamus. J. Neurosci., 18: 3489–3500. Kiss, J., Csaba, Z., Csaki, A. and Halasz, B. (2005) Glutamatergic innervation of neuropeptide Y and pro-opiomelanocortin-containing neurons in the hypothalamic arcuate nucleus of the rat. Eur. J. Neurosci., 21: 2111–2119. Kristensen, P., Judge, M.E., Thim, L., Ribel, U., Christjansen, K.N., Wulff, B.S., Clausen, J.T., Jensen, P.B., Madsen, O.D., Vrang, N., Larsen, P.J. and Hastrup, S. (1998) Hypothalamic CART is a new anorectic peptide regulated by leptin. Nature, 393: 72–76. Lam, T.K., Schwartz, G.J. and Rossetti, L. (2005) Hypothalamic sensing of fatty acids. Nat. Neurosci., 8: 579–584. Legradi, G. and Lechan, R.M. (1999) Agouti-related protein containing nerve terminals innervate thyrotropin-releasing hormone neurons in the hypothalamic paraventricular nucleus. Endocrinology, 140: 3643–3652. Levin, B.E., Routh, V.H., Kang, L., Sanders, N.M. and DunnMeynell, A.A. (2004) Neuronal glucosensing: what do we know after 50 years? Diabetes, 53: 2521–2528. Llinas, R. and Jahnsen, H. (1982) Electrophysiology of mammalian thalamic neurones in vitro. Nature, 297: 406–408. Llinas, R. and Yarom, Y. (1981) Electrophysiology of mammalian inferior olivary neurones in vitro. Different types of voltage-dependent ionic conductances. J. Physiol., 315: 549–567. Luther, J.A., Halmos, K.C. and Tasker, J.G. (2000) A slow transient potassium current expressed in a subset of neurosecretory neurons of the hypothalamic paraventricular nucleus. J. Neurophysiol., 84: 1814–1825. Magee, J.C. (1998) Dendritic hyperpolarization-activated currents modify the integrative properties of hippocampal CA1 pyramidal neurons. J. Neurosci., 18: 7613–7624. Magee, J.C. (1999) Dendritic lh normalizes temporal summation in hippocampal CA1 neurons. Nat. Neurosci., 2: 508–514. Marcus, J.N., Aschkenasi, C.J., Lee, C.E., Chemelli, R.M., Saper, C.B., Yanagisawa, M. and Elmquist, J.K. (2001) Differential expression of orexin receptors 1 and 2 in the rat brain. J. Comp. Neurol., 435: 6–25.

Mercer, J.G., Hoggard, N., Williams, L.M., Lawrence, C.B., Hannah, L.T. and Trayhurn, P. (1996) Localization of leptin receptor mRNA and the long form splice variant (Ob-Rb) in mouse hypothalamus and adjacent brain regions by in situ hybridization. FEBS Lett., 387: 113–116. Mizuno, T.M., Kleopoulos, S.P., Bergen, H.T., Roberts, J.L., Priest, C.A. and Mobbs, C.V. (1998) Hypothalamic pro-opiomelanocortin mRNA is reduced by fasting and [corrected] in ob/ob and db/db mice, but is stimulated by leptin. Diabetes, 47: 294–297. Mizuno, T.M. and Mobbs, C.V. (1999) Hypothalamic agoutirelated protein messenger ribonucleic acid is inhibited by leptin and stimulated by fasting. Endocrinology, 140: 814–817. Morton, G.J. and Schwartz, M.W. (2001) The NPY/AgRP neuron and energy homeostasis. Int. J. Obes. Relat. Metab. Disord., 25(Suppl 5): S56–S62. Munzberg, H. and Myers, M.G. (2005) Molecular and anatomical determinants of central leptin resistance. Nat. Neurosci., 8: 566–570. Niswender, K.D., Morrison, C.D., Clegg, D.J., Olson, R., Baskin, D.G., Myers Jr., M.G., Seeley, R.J. and Schwartz, M.W. (2003) Insulin activation of phosphatidylinositol 3kinase in the hypothalamic arcuate nucleus: a key mediator of insulin-induced anorexia. Diabetes, 52: 227–231. Niswender, K.D., Morton, G.J., Stearns, W.H., Rhodes, C.J., Myers Jr., M.G. and Schwartz, M.W. (2001) Intracellular signalling. Key enzyme in leptin-induced anorexia. Nature, 413: 794–795. Niswender, K.D. and Schwartz, M.W. (2003) Insulin and leptin revisited: adiposity signals with overlapping physiological and intracellular signaling capabilities. Front. Neuroendocrinol., 24: 1–10. Obici, S., Feng, Z., Karkanias, G., Baskin, D.G. and Rossetti, L. (2002) Decreasing hypothalamic insulin receptors causes hyperphagia and insulin resistance in rats. Nat. Neurosci., 5: 566–572. Obici, S., Feng, Z., Morgan, K., Stein, D., Karkanias, G. and Rossetti, L. (2002) Central administration of oleic acid inhibits glucose production and food intake. Diabetes, 51: 271–275. Obici, S. and Rossetti, L. (2003) Minireview: nutrient sensing and the regulation of insulin action and energy balance. Endocrinology, 144: 5172–5178. Obici, S., Zhang, B.B., Karkanias, G. and Rossetti, L. (2002) Hypothalamic insulin signaling is required for inhibition of glucose production. Nat. Med., 8: 1376–1382. Pape, H.C. (1996) Queer current and pacemaker: the hyperpolarization-activated cation current in neurons. Annu. Rev. Physiol., 58: 299–327. Peyron, C., Tighe, D.K., van den Pol, A.N., de Lecea, L., Heller, H.C., Sutcliffe, J.G. and Kilduff, T.S. (1998) Neurons containing hypocretin (orexin) project to multiple neuronal systems. J. Neurosci., 18: 9996–10015. Phillips, M.S., Liu, Q., Hammond, H.A., Dugan, V., Hey, P.J., Caskey, C.J. and Hess, J.F. (1996) Leptin receptor missense mutation in the fatty Zucker rat. Nat. Genet., 13: 18–19.

154 Pinto, S., Roseberry, A.G., Liu, H., Diano, S., Shanabrough, M., Cai, X., Friedman, J.M. and Horvath, T.L. (2004) Rapid rewiring of arcuate nucleus feeding circuits by leptin. Science, 304: 110–115. Pocai, A., Lam, T.K., Gutierrez-Juarez, R., Obici, S., Schwartz, G.J., Bryan, J., Aguilar-Bryan, L. and Rossetti, L. (2005) Hypothalamic K(ATP) channels control hepatic glucose production. Nature, 434: 1026–1031. Poulain, D.A. and Wakerley, J.B. (1982) Electrophysiology of hypothalamic magnocellular neurones secreting oxytocin and vasopressin. Neuroscience, 7: 773–808. Poulain, P., Decrocq, N. and Mitchell, V. (2003) Direct inhibitory action of galanin on hypothalamic arcuate nucleus neurones expressing galanin receptor Gal-r1 mRNA. Neuroendocrinology, 78: 105–117. Qu, D., Ludwig, D.S., Gammeltoft, S., Piper, M., Pelleymounter, M.A., Cullen, M.J., Mathes, W.F., Przypek, R., Kanarek, R. and Maratos-Flier, E. (1996) A role for melanin-concentrating hormone in the central regulation of feeding behaviour. Nature, 380: 243–247. Rogawski, M.A. (1985) The A-current: how ubiquitous a feature of excitable cells is it? Trends Neurosci., 8: 214–219. Roseberry, A.G., Liu, H., Jackson, A.C., Cai, X. and Friedman, J.M. (2004) Neuropeptide Y-mediated inhibition of proopiomelanocortin neurons in the arcuate nucleus shows enhanced desensitization in ob/ob mice. Neuron, 41: 711–722. Rosin, D.L., Weston, M.C., Sevigny, C.P., Stornetta, R.L. and Guyenet, P.G. (2003) Hypothalamic orexin (hypocretin) neurons express vesicular glutamate transporters VGLUT1 or VGLUT2. J. Comp. Neurol., 465: 593–603. Schwartz, M.W., Seeley, R.J., Woods, S.C., Weigle, D.S., Campfield, L.A., Burn, P. and Baskin, D.G. (1997) Leptin increases hypothalamic pro-opiomelanocortin mRNA expression in the rostral arcuate nucleus. Diabetes, 46: 2119–2123. Shimada, M., Tritos, N.A., Lowell, B.B., Flier, J.S. and Maratos-Flier, E. (1998) Mice lacking melanin-concentrating hormone are hypophagic and lean. Nature, 396: 670–674. Shimokawa, I., Fukuyama, T., Yanagihara-Outa, K., Tomita, M., Komatsu, T., Higami, Y., Tuchiya, T., Chiba, T. and Yamaza, Y. (2003) Effects of caloric restriction on gene expression in the arcuate nucleus. Neurobiol. Aging, 24: 117–123. Shuto, Y., Shibasaki, T., Otagiri, A., Kuriyama, H., Ohata, H., Tamura, H., Kamegai, J., Sugihara, H., Oikawa, S. and Wakabayashi, I. (2002) Hypothalamic growth hormone secretagogue receptor regulates growth hormone secretion, feeding, and adiposity. J. Clin. Invest., 109: 1429–1436. Spanswick, D., Smith, M.A., Groppi, V.E., Logan, S.D. and Ashford, M.L. (1997) Leptin inhibits hypothalamic neurons by activation of ATP-sensitive potassium channels. Nature, 390: 521–525.

Spanswick, D., Smith, M.A., Mirshamsi, S., Routh, V.H. and Ashford, M.L. (2000) Insulin activates ATP-sensitive K+ channels in hypothalamic neurons of lean, but not obese rats. Nat. Neurosci., 3: 757–758. Takahashi, K.A. and Cone, R.D. (2005) Fasting induces a large, leptin-dependent increase in the intrinsic action potential frequency of orexigenic arcuate nucleus neuropeptide Y/ Agouti-related protein neurons. Endocrinology, 146: 1043–1047. Tasker, J.G. and Dudek, F.E. (1991) Electrophysiological properties of neurones in the region of the paraventricular nucleus in slices of rat hypothalamus. J. Physiol., 434: 271–293. Thomzig, A., Laube, G., Pruss, H. and Veh, R.W. (2005) Pore-forming subunits of K-ATP channels, Kir6.1 and Kir6.2, display prominent differences in regional and cellular distribution in the rat brain. J. Comp. Neurol., 484: 313–330. van den Top, M., Lee, K., Whyment, A.D., Blanks, A.M. and Spanswick, D. (2004) Orexigen-sensitive NPY/AgRP pacemaker neurons in the hypothalamic arcuate nucleus. Nat. Neurosci., 7: 493–494. van Houten, M., Posner, B.I., Kopriwa, B.M. and Brawer, J.R. (1980) Insulin-binding sites localized to nerve terminals in rat median eminence and arcuate nucleus. Science, 207: 1081–1083. Wang, R., Liu, X., Hentges, S.T., Dunn-Meynell, A.A., Levin, B.E., Wang, W. and Routh, V.H. (2004) The regulation of glucose-excited neurons in the hypothalamic arcuate nucleus by glucose and feeding-relevant peptides. Diabetes, 53: 1959–1965. Whyment, A.D., Wilson, J.M., Renaud, L.P. and Spanswick, D. (2004) Activation and integration of bilateral GABA-mediated synaptic inputs in neonatal rat sympathetic preganglionic neurones in vitro. J. Physiol., 555: 189–203. Willesen, M.G., Kristensen, P. and Romer, J. (1999) Co-localization of growth hormone secretagogue receptor and NPY mRNA in the arcuate nucleus of the rat. Neuroendocrinology, 70: 306–316. Woods, S.C., Chavez, M., Park, C.R., Riedy, C., Kaiyala, K., Richardson, R.D., Figlewicz, D.P., Schwartz, M.W., Porte Jr., D. and Seeley, R.J. (1996) The evaluation of insulin as a metabolic signal influencing behavior via the brain. Neurosci. Biobehav. Rev., 20: 139–144. Xu, A.W., Kaelin, C.B., Takeda, K., Akira, S., Schwartz, M.W. and Barsh, G.S. (2005) PI3K integrates the action of insulin and leptin on hypothalamic neurons. J. Clin. Invest., 115: 951–958. Yamanaka, A., Kunii, K., Nambu, T., Tsujino, N., Sakai, A., Matsuzaki, I., Miwa, Y., Goto, K. and Sakurai, T. (2000) Orexin-induced food intake involves neuropeptide Y pathway. Brain Res., 859: 404–409.