Leptin directly regulate intrinsic neuronal excitability in hypothalamic POMC neurons but not in AgRP neurons in food restricted mice

Accepted Manuscript Title: Leptin directly regulate intrinsic neuronal excitability in hypothalamic POMC neurons but not in AgRP neurons in food restricted mice Authors: Soojung Lee, Jooyoung Lee, Gil Myoung Kang, Min-Seon Kim PII: DOI: Reference:

S0304-3940(18)30392-6 https://doi.org/10.1016/j.neulet.2018.05.041 NSL 33620

To appear in:

Neuroscience Letters

Received date: Revised date: Accepted date:

6-7-2015 9-5-2018 28-5-2018

Please cite this article as: Soojung Lee, Jooyoung Lee, Gil Myoung Kang, MinSeon Kim, Leptin directly regulate intrinsic neuronal excitability in hypothalamic POMC neurons but not in AgRP neurons in food restricted mice, Neuroscience Letters https://doi.org/10.1016/j.neulet.2018.05.041 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Leptin directly regulate intrinsic neuronal excitability in hypothalamic POMC neurons but not in AgRP neurons in food restricted mice

Soojung Leea*, Jooyoung Leea, Gil Myoung Kangb, Min-Seon Kimb

Department of Oral Physiology, Faculty of Dentistry, Kyung Hee University, Seoul, Korea b Asan

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Institute for Life Science, University of Ulsan College of Medicine, Seoul, Korea

* Corresponding author at: Department of Oral Physiology, Faculty of Dentistry, Kyung Hee

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University, Seoul 130-701, Korea.

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Tel/Fax: +82 2 961 0824

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Email address: [email protected] (Soojung Lee, Ph. D.)

Hightlights

Leptin increased firing frequencies of POMC neurons after food deprivation.

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Leptin did not affect neuronal excitability of AgRP neurons after food deprivation.

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Leptin decreased spontaneous inhibitory synaptic inputs to POMC neurons.

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ABSTRACT

Leptin plays a pivotal role in the central control of energy balance through leptin receptors expressed on specific hypothalamic nuclei. Leptin suppresses food intake and body weight and ameliorates hyperglycemia by acting on the AgRP and POMC neurons of the arcuate nucleus. Leptin action on

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POMC neurons are essential for control of body weight and blood glucose levels and are known to be mediated by JAK-STAT3 and PI3K signalling pathway thus increase POMC mRNA and intrinsic

hyperpolarize AgRP neurons through change of K+ conductance.

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excitability. The effects of leptin on AgRP neurons are not as clear although it has been reported to

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Using cell-attached patch and whole cell patch configuration, we directly assessed neuronal response

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to leptin in GFP labelled AgRP or POMC neurons in mice after 18 hour of food deprivation. We

However, leptin does not have any effect on intrinsic

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decreased inhibitory synaptic inputs.

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found leptin has a direct effect on POMC neuron through increased intrinsic excitability and

excitability of AgRP neurons in fasted condition although food deprivation induced increase of firing

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frequency of AgRP neurons. In conclusion, leptin probably has a direct and acute effect on POMC neurons but not on AgRP neurons to control their excitability within feeding-regulatory neuronal

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Keywords:

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circuitry.

AgRP

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POMC Leptin

Arcuate nucleus Neuronal excitability Food deprivation

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Abbreviations: Arc, arcuate nucleus; Vm, membrane potential; PI3K, phosphatidylinositol 3-kinases;

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ACSF, artificial cerebrospinal fluid; AgRP, agouti-related protein; POMC, proopiomelanocortin

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1. Introduction

Hypothalamus, situated in the mediobasal part of the brain has a determining role in energy balance to

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such as insulin, leptin or glucose. Arcuate nucleus (Arc), located privileged next to the third ventricle

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has been postulated to have a fundamental role in sensing the global energy status from the body and thus act as a first sensor of peripheral signals [5]. Arc contains special neuronal populations, neurons

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expressing POMC and CART that provide anorexigenic effect, neurons releasing AgRP and NPY that exert orexigenic effect. In the situation of negative energy balance or fasting, the expression of

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AgRP is increased and POMC expression is decreased [3, 6], and baseline firing frequency of AgRP

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neurons is increased [19].

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The endocrine hormone leptin derived from white adipose tissue plays a pivotal role in the adaptive

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response of the central control of energy homeostasis through leptin receptors expressed on specific hypothalamic nuclei [10, 13, 23]. Leptin regulate food intake and body weight and ameliorate glucose

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metabolism [21]. Leptin receptor as well as insulin receptor was expressed particularly highly in the AgRP and POMC neurons of the Arc [22, 23]. Numerous studies indicated that leptin depolarize and activate POMC neurons via two different signalling pathways, nuclear translocation of the

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transcription factor, signal transducer and activator of transcription 3 (STAT3) and possibly

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phosphatidylinositol 3-kinases (PI3K), thus generate anorexigenic signal mediated by α-melanocyte stimulating hormone [1, 8, 23]. It is well known that POMC neurons are essential in mediating leptin

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actions in the brain. It has been reported that leptin depolarizes membrane potential (Vm) of POMC neuron and increases the firing rate of POMC neurons by activating nonspecific cation channels [8, 12, 23, 25]. PI3K activation has been reported mediating this effect but exact molecular mechanism has yet to be fully elucidated.

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Meanwhile, the effects of leptin on AgRP neurons are not as clear, although reports has suggested leptin acutely decrease intrinsic excitability by hyperpolarizing AgRP neurons through activation of ATP-dependent potassium (KATP) channels or change of K+ conductance [2, 7, 22] but others reported leptin did not change Vm or firing frequency or PI3K activation in AgRP neuron [4, 25]. Besides, although electrical activity were presumed to determine neurotransmitter /neuropeptide release and

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physiological output of neuron, there are reports that electrical activity and secretion are not necessarily coupled [9, 15].

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Therefore it is important to clarify leptin’s effect on neuronal excitability in AgRP and POMC neurons and determine which neurons among AgRP and POMC neurons are the primary targets for

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an important anorexigenic hormone, leptin.

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2. Material and Methods

2.1 Animals Mice with GFP labelling in their POMC neurons and AgRP neurons were generated by mating POMC-cre mice or AgRP-Ires-cre mice (both from Dr. Joel K. Elmquist, University of Texas

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Southwestern Medical Center) with mice carry a floxed STOP cassette upstream of an enhanced green

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fluorescence protein (eGFP) (Jackson laboratory, Maine, MA).

2.2 Slice preparation

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All experiments were performed on 5-8 weeks of either either sex of Animal care and use conformed

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to the institutional guidelines of the Asan Institute for Life Science (Seoul, Korea).

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After 18 hours of overnight food deprivation, animals were deeply anaesthetised with halothane,

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decapitated and the brain was removed quickly and placed in ice-cold cutting solution that contained (in mM): 250 Sucrose, 26 NaHCO3, 10 D(+)‐Glucose, 4 MgCl2, 3 myo‐inositol, 2.5 KCl, 2 Sodium

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pyruvate, 1.25 NaH2PO4, 0.5 Ascorbic acid 0.1 CaCl2, and 1 Kynurenic acid, pH 7.4. All solution was gassed with 95% O2‐5% CO2. 250 μM thick coronal slices were cut using vibratome (Leica VTS 1200) and transferred to extracellular ACSF solution: 126 NaCl, 24 NaHCO3, 1 NaH2PO4, 2.5 KCl,

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2.5 CaCl2, 2 MgCl2, 10 D(+)‐Glucose, and 0.4 Ascorbic acid, pH 7.4. After collecting two slices that

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contain Arc, they were incubated at room temperature for at least one hour prior to recording. Most of recordings were performed within 3 hours after cutting. 1-2 data were collected maximally from

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each animal. Slice was transferred to a recording chamber perfused continuously with ASCF solution (flow rate; 1.5 ml/min). Slice chamber was mounted on the stage of an upright Olympus microscope and viewed with an X40 water immersion objective with infrared differential interference contrast optics. Location of Arc was easily identified with third ventricle and median eminence. Target

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neurons were identified with band pass filtered (465–495 nm) green fluorescence (CL-2005, CrystaLaser) and image was visualized by CCD camera (MCE-B013-U, Mightex Systems).

2.3 Electrophysiology For loose cell-attached or whole-cell patch recordings from Arc neurons, electrodes (4-6 MΩ) were

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filled with a ACSF or a solution containing (in mM): 130 K-gluconate, 2 NaCl, 4 MgCl2, 20 HEPES, 4 MgATP, 0.4 Na2GTP, 0.5 EGTA, 10 Na2Phosphocreatine (pH 7.3, Osmol 290-295). Access

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resistance for cell-attached and whole cell patch recordings were 100-120 MΩ and 4-6 MΩ, respectively. All reagents were purchased from Sigma unless otherwise indicated. Leptin was

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purchased from Phoenix Pharmaceuticals. Recordings lasted for more than 30 mins were selected for

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analysis. For whole-cell recordings for IPSCs, pipettes were filled with a solution containing (in mM):

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140 CsCl, 10 HEPES, 5 MgCl2, 1 BAPTA, 5 MgATP, 0.3 MgGTP, 10 Na2Phosphocreatine, 10 QX314 (pH 7.3, Osmol 290-295). IPSCs were recorded under voltage clamp with a holding potential of

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-60 mV. Electrical signals were digitized and sampled at 50 μs intervals with Digidata 1440A and

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Multiclamp 700B amplifier (Molecular Devices) using pCLAMP 10.4 sofware (Molecular Devices). Data were filtered at 2 kHz. All recordings were recorded in gap free mode continuously.

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2.4 Data analysis

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Off‐line analysis was carried out using Clampfit 10.4, Minianalysis (Synaptosoft), OriginPro 8.5(OriginLab) and Excel (Microsoft) software. Numerical data were presented as means ± s.e.m.

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Each data point was obtained from averaged firing frequency of every 2 minutes. The significance of data for comparison was assessed by Student’s two‐tailed unpaired t‐test and significance level was displayed as * (p < 0.05), ** (p < 0.01), *** (p < 0.005).

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3. Results Cell attached patch recording was attempted in AgRP-GFP or POMC-GFP neurons in Arc prepared from mice with/without 18 hours of food deprivation(fasted/fed mice). Baseline recording was typically acquired for 6-10 mins after stabilization of firing frequency. In fed mice, the basal spike frequency of Arc AgRP neurons was very low (0.97±0.15 Hz, n=6, Fig. 1A) compared to mean basal

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spike frequency of POMC neurons (1.84±0.31 Hz, n=10, Fig. 1B). After 18 hours of food deprivation, spontaneous firings of AGRP neuron were significantly increased about three times (3.11±0.45 Hz,

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n=16, p <0.01, student unpaired t-test, Fig. 1A), whereas firing of POMC neuron did not change (2.16±0.42 Hz, n=12, Fig. 1B).

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Leptin (100 nM) was bath applied by perfusion and neuronal firing frequency was constantly

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monitored in the slice from food deprived mouse. Recordings lasted for more than 30 mins were selected for analysis. Remarkably, spike frequency of AgRP neuron did not change after leptin

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treatment (3.00±0.48 Hz vs 3.37±0.53 Hz, n=8, Fig. 2AB). However, on the contrary to leptin’s effect on AgRP, leptin application markedly increase spike frequency of POMC neurons. Increase of firing

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frequency was significantly higher after 30mins of leptin application (2.55±0.54 Hz vs 4.63±0.69 Hz, n=7, p < 0.05, Fig. 2CD).

As previous studies have reported that leptin acutely hyperpolarized Vm and decreased firing

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frequency of AgRP neurons [2, 8], we tried whole cell recording which contained ATP/GTP in

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intracellular solution as same as studies mentioned above to resolve discrepancies. Whole cell recording was carried out on AGRP-GFP neuron from mice after 18 hours food deprivation. Baseline

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spike frequency from whole cell recording was slightly lower than that from cell-attached patch recording (3.00±0.48 Hz vs 2.39±0.48 Hz, Fig. 2B and Fig. 3B) due to the high concentration of potassium in electrode but it was not significant. In contrast to no effect of leptin on firing of AgRP neurons in cell attached configuration, leptin application quickly hyperpolarized resting membrane potential of AGRP neurons in whole cell

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recording (-44.86±0.86 mV vs -51.88±0.53, n=4, p < 0.05) and dramatically decreased spike frequency even after 10 mins-application of leptin (2.39±0.48 Hz vs 0.39±0.21 Hz, p < 0.01). After 30 mins-application of leptin, spontaneous firing was almost abolished (2.39±0.48 Hz vs 0.16±0.12 Hz, p < 0.005, Fig. 3B). As previous reports demonstrated that inhibitory synaptic inputs but not excitatory synaptic inputs

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are major targets for leptin effects on POMC neuron [11, 14], we next evaluated if there are altered GABAergic tones to POMC neurons after 18 hours food deprivation. We found that fasting produced

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significant increase of spontaneous IPSCs in POMC neurons (4.01±0.63 Hz vs 7.00±0. 96 Hz, n=12 vs n=7, p < 0.05). Leptin treatment for 30mins markedly decreased fasting mediated sIPSCs increase in POMC neurons (7.48±1.26 Hz vs 3.66±0. 66 Hz, n=4, p < 0.05, Fig 4B) which was consistent with

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the view that increased inhibitory tone in fasting state was leptin sensitive[24]. Although leptin’s

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effects on sIPSCs are obvious in POMC neurons, whether they have definitive effects on neuronal

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excitability is not clear as fasting mediated increased inhibitory tone seems to be ineffective to modulate spontaneous firing frequency of POMC neurons. It is worth to refer to previous reports that

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fasting potently upregulated AgRP mRNA levels but only slightly increased POMC mRNA[5]. Whether leptin induced increased excitability of POMC neuron are due to decreased GABAergic

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tones or K+ conductance change mediated by PI3K activation needs further clarified.

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4. Discussion . Many previous electrophysiological studies suggested that leptin’s direct modulation on intrinsic excitability and resulting reduced firing frequency of AgRP neurons can be attributable to decreased feeding [2, 8, 18, 26]. Surprisingly, our finding showed, after overnight food deprivation scheme, that

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leptin does not have any direct effect on intrinsic excitability of AgRP neurons compared to depolarizing effect on POMC neurons with our cell-attached patch configuration. The discordant

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findings about leptin’s effect on AgRP neuron may be due to different experimental conditions as studies above were carried out by whole cell patch recording with electrode prefilled with solution to mimic ATP/GTP concentration. Dialysis of unknown cytosolic component and high concentration of

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ATP than pre-experimental condition after fasting may have affected KATP channel sensitivity and K+

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conductance of other channels or inhibit AMP-activated protein kinase that acutely modulate neuronal excitability [3, 4, 20]. Our whole cell recording on AgRP neuron confirmed that leptin

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rapidly hyperpolarized Vm and decreased firing frequency, indicating that leptin’s hyperpolarizing

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effect through PI3K activation is indeed induced by experimental conditions. A couple of studies have indicated that leptin did not alter membrane potential and firing frequency under perforated patch recording condition that does not disturb internal milieu of AgRP neuron [3,

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4]. Leptin also was indicated that it did not translocate PI3K to the membrane in AgRP neuron but

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activated PI3K and translocated to the membrane in POMC neuron [25]. Leptin depolarized Vm through nonselective cation channels [25] as PI3K plays a central role to modulate ion channels in

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vitro . Our finding is in accord with current views of leptin effects on POMC neuron. Leptin regulation of cellular function is mediated by two different pathways, the activation of Janus-activated kinase (JAK)/STAT3 and activation of PI3K pathways. Although both pathways seems to be activated by leptin independently, activation of PI3K signalling rather than STAT3 are essential for controlling neuronal firing and acute anorexigenic effect of leptin in POMC neuron [12].

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Interestingly, it has been reported that withdrawal of leptin translocate PI3K in AgRP neuron but this effect needs presynaptic neurotransmitter release or synaptic inputs [25]. The finding that insulin translocates PI3K and depolarize Vm and increase firing of AgRP neuron [4, 25] suggest that leptin withdrawal may induce increase of AgRP neuron firing. Leptin’s inhibitory effect on PI3K activation

essential for leptin’s inhibitory role for PI3K pathway in AgRP neuron.

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requires synaptic transmission implicated a possible role for NMDA receptor activation that might be

In our study, another consideration for determinants for neuronal excitability is the level of glucose

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in Arc after fasting. Physiological hypothalamic glucose levels vary from 0.7 to 2.5 mM between the fasted and fed state. At baseline, blood glucose level was reported about 5 ~ 8 mM in euglycemia and

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ventromedial nucleus glucose levels never exceeded 4.5 mM even blood glucose level was increased

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to 20mM [16, 17]. The majority of in vitro studies of glucose sensing neurons still used standard

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ACSF solutions containing 10-20 mM glucose. In fed condition, Arc and ventromedial nucleus

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glucose levels were similar ( 20% of blood glucose level) and there were distinct diffusion barriers between them [9]. However, during fasting and glucodeprivation, structural component of this barrier

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was altered and Arc glucose levels significantly surpass glucose level in the ventromedial nucleus [16]. Due to the privileged routes for glucose diffusion to Arc but not to ventromedial nucleus, glucose level of Arc neuron may not be affected much by fasting state. Claret et al., showed when glucose

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was reduced from 2 to 0.1 mM a minority of mouse AgRP neurons were hyperpolarized but with 2-

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10 mM concentration of glucose, membrane potential and firing frequency of AgRP neuron was not changed [4]. Considering these findings, our 10mM glucose level may not have affected the firing

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frequency or intrinsic excitability of Arc neuron in our glucose concentration. In conclusion, we showed that leptin has a direct and acute effect on neuronal excitability on POMC neurons but not on AgRP neurons but further study needed for leptin’s indirect network or prolonged effect on AgRP neurons in the future.

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Conflict of interest There is no conflict of interest concerning the authors in conducting this study and preparing this manuscript.

Contributors

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SL designed and conducted the experiment, analysed the data, and wrote the manuscript. JL and GML

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conducted the experiment. MSK wrote the manuscript.

Acknowledgments

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This work was supported by the National Research Foundation of Korea (NRF) grant funded by the

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Korean Government (MEST) (No.2012R1A5A2051385) and the Grant from Kyung Hee University

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in 2012 (KHU-20120485).

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References [1] N. Balthasar, R. Coppari, J. McMinn, S.M. Liu, C.E. Lee, V. Tang, C.D. Kenny, R.A. McGovern, S.C. Chua Jr, J.K. Elmquist, B.B. Lowell, Leptin Receptor Signaling in POMC Neurons Is Required for Normal Body Weight Homeostasis, Neuron 42 (2004) 983-991. [2] S.B. Baver, K. Hope, S. Guyot, C. Bjørbaek, C. Kaczorowski, K.M.S. O'Connell, Leptin Modulates the Intrinsic Excitability of AgRP/NPY Neurons in the Arcuate Nucleus of the Hypothalamus, The Journal of Neuroscience 34 (2014) 5486-5496. [3] B.F. Belgardt, T. Okamura, J.C. Brüning, Hormone and glucose signalling in POMC and AgRP neurons, The Journal of Physiology 587 (2009) 5305-5314. [4] M. Claret, M.A. Smith, R.L. Batterham, C. Selman, A.I. Choudhury, L.G.D. Fryer, M. Clements, H. Al-Qassab, H. Heffron, A.W. Xu, J.R. Speakman, G.S. Barsh, B. Viollet, S. Vaulont, M.L.J. Ashford, D. Carling, D.J. Withers, AMPK is essential for energy homeostasis regulation and glucose sensing by POMC and AgRP neurons, The Journal of Clinical Investigation 117 (2007) 2325-2336. [5] R.D. Cone, Anatomy and regulation of the central melanocortin system, Nat Neurosci 8 (2005) 571-578. [6] R.D. Cone, M.A. Cowley, A.A. Butler, W. Fan, M.D. L, M.J. Low, The arcuate nucleus as a conduit for diverse signals relevant to energy homeostasis, International Journal of Obesity 25 (2001) S63-S67. [7] M.A. Cowley, R.D. Cone, P. Enriori, I. Louiselle, S.M. Williams, A.E. Evans, Electrophysiological Actions of Peripheral Hormones on Melanocortin Neurons, Annals of the New York Academy of Sciences 994 (2003) 175-186. [8] M.A. Cowley, J.L. Smart, M. Rubinstein, M.G. Cerdan, S. Diano, T.L. Horvath, R.D. Cone, M.J. Low, Leptin activates anorexigenic POMC neurons through a neural network in the arcuate nucleus, Nature 411 (2001) 480-484. [9] A.A. Dunn-Meynell, N.M. Sanders, D. Compton, T.C. Becker, J.-i. Eiki, B.B. Zhang, B.E. Levin, Relationship among Brain and Blood Glucose Levels and Spontaneous and Glucoprivic Feeding, The Journal of Neuroscience 29 (2009) 7015-7022. [10] M.V. Houten, B.I. Posner, B.M. Kopriwa, J.R. Brawer, Insulin-Binding Sites in the Rat Brain: In Vivo Localization to the Circumventricular Organs by Quantitative Radioautography, Endocrinology 105 (1979) 666-673. [11] T. Liu, D. Kong, Bhavik P. Shah, C. Ye, S. Koda, A. Saunders, Jun B. Ding, Z. Yang, Bernardo L. Sabatini, Bradford B. Lowell, Fasting Activation of AgRP Neurons Requires NMDA Receptors and Involves Spinogenesis and Increased Excitatory Tone, Neuron 73 (2012) 511-522. [12] C.D. Morrison, G.J. Morton, K.D. Niswender, R.W. Gelling, M.W. Schwartz, Leptin inhibits hypothalamic Npy and Agrp gene expression via a mechanism that requires phosphatidylinositol 3-OH-kinase signaling, Vol. 289, 2005, E1051-E1057 pp. [13] M.G. Myers Jr, R.L. Leibel, R.J. Seeley, M.W. Schwartz, Obesity and leptin resistance: distinguishing cause from effect, Trends in Endocrinology & Metabolism 21 (2010) 643-651. [14] S. Pinto, A.G. Roseberry, H. Liu, S. Diano, M. Shanabrough, X. Cai, J.M. Friedman, T.L. Horvath, Rapid Rewiring of Arcuate Nucleus Feeding Circuits by Leptin, Science 304 (2004) 110-115. [15] L. Plum, X. Ma, B. Hampel, N. Balthasar, R. Coppari, xFc, H. nzberg, M. Shanabrough, D. Burdakov, E. Rother, R. Janoschek, J. Alber, B.F. Belgardt, L. Koch, J. Seibler, F. Schwenk, C. Fekete, A. Suzuki, T.W. Mak, W. Krone, T.L. Horvath, F.M. Ashcroft, Br, xFc, J.C. ning, Enhanced PIP3 signaling in POMC neurons causes KATP channel activation and leads to diet-sensitive obesity, The Journal of Clinical Investigation 116 (2006) 1886-1901. [16] V. Routh, L. Hao, A.M. Santiago, Z. Sheng, C. Zhou, Hypothalamic glucose sensing: making ends meet, Frontiers in Systems Neuroscience 8 (2014).

13

[20]

[21]

[22]

[23]

A

CC

EP

TE D

[26]

M

[25]

A

N

[24]

IP T

[19]

SC R

[18]

V.H. Routh, Glucose-sensing neurons: Are they physiologically relevant?, Physiology & Behavior 76 (2002) 403-413. D. Spanswick, M.A. Smith, V.E. Groppi, S.D. Logan, M.L.J. Ashford, Leptin inhibits hypothalamic neurons by activation of ATP-sensitive potassium channels, Nature 390 (1997) 521-525. K.A. Takahashi, R.D. Cone, Fasting Induces a Large, Leptin-Dependent Increase in the Intrinsic Action Potential Frequency of Orexigenic Arcuate Nucleus Neuropeptide Y/AgoutiRelated Protein Neurons, Endocrinology 146 (2005) 1043-1047. N. Teramoto, T. Tomoda, T. Yunoki, Y. Ito, Different glibenclamide-sensitivity of ATPsensitive K+ currents using different patch-clamp recording methods, European Journal of Pharmacology 531 (2006) 34-40. E. van de Wall, R. Leshan, A.W. Xu, N. Balthasar, R. Coppari, S.M. Liu, Y.H. Jo, R.G. MacKenzie, D.B. Allison, N.J. Dun, J. Elmquist, B.B. Lowell, G.S. Barsh, C. de Luca, M.G. Myers, G.J. Schwartz, S.C. Chua, Collective and Individual Functions of Leptin Receptor Modulated Neurons Controlling Metabolism and Ingestion, Endocrinology 149 (2008) 17731785. M. van den Top, K. Lee, A.D. Whyment, A.M. Blanks, D. Spanswick, Orexigen-sensitive NPY/AgRP pacemaker neurons in the hypothalamic arcuate nucleus, Nat Neurosci 7 (2004) 493-494. L. Varela, T.L. Horvath, Leptin and insulin pathways in POMC and AgRP neurons that modulate energy balance and glucose homeostasis, Vol. 13, 2012, 1079-1086 pp. L. Vong, C. Ye, Z. Yang, B. Choi, S. Chua Jr, Bradford B. Lowell, Leptin Action on GABAergic Neurons Prevents Obesity and Reduces Inhibitory Tone to POMC Neurons, Neuron 71 (2011) 142-154. A.W. Xu, C.B. Kaelin, K. Takeda, S. Akira, M.W. Schwartz, G.S. Barsh, PI3K integrates the action of insulin and leptin on hypothalamic neurons, The Journal of Clinical Investigation 115 (2005) 951-958. M.-J. Yang, F. Wang, J.-H. Wang, W.-N. Wu, Z.-L. Hu, J. Cheng, D.-F. Yu, L.-H. Long, H. Fu, N. Xie, J.-G. Chen, PI3K integrates the effects of insulin and leptin on large-conductance Ca2+-activated K+ channels in neuropeptide Y neurons of the hypothalamic arcuate nucleus, Vol. 298, 2010, E193-E201 pp.

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Figure legends Fig. 1. Baseline spontaneous firing frequencies of arcuate nucleus neurons with or without 18 h food restriction. (A) Mean firing frequencies (±s.e.m.) of AgRP neurons were recorded in cell-attached patch configuration using hypothalamic slice preparations from either AgRP-GFP mice in fed or fasted state. Fasting dramatically increased firing frequency of AgRP neuron (** p < 0.01, paired ttest). (B) Mean firing frequencies of POMC neurons from POMC-GFP mice in fed or fasted state (p > 0.5). In all electrophysiological recordings, each n represent independent neuron and slice and no more than two slices were used per animal.

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Fig. 2. Leptin action on spontaneous firing frequencies of arcuate nucleus neurons from fasted mice. (A) Upper panel showed time-course of bath application of leptin (100nM) on recorded neuron. Lower panel showed representative firing responses of an AgRP neuron recorded in cell-attached mode. (B) The bar graph represents mean±s.e.m. after 30 mins of leptin application (p > 0.5). (C) The representative firing responses of a POMC neuron recorded in cell-attached mode. (D) The bar graph represents mean±s.e.m. after 30 mins of leptin application (* p < 0.05, paired t-test).

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Fig. 3. Leptin action on spontaneous firing frequencies of AgRP neuron from fasted mice in wholecell patch configuration. (A) Upper panel showed same time-course of bath application of leptin(100nM) on recorded neuron as cell attached patch mode. Lower panel showed representative firing responses and resting membrane potential of an AgRP neuron recorded in whole-cell mode. (B) The bar graph represents mean±s.e.m. after 30 mins of leptin application (*** p < 0.005, paired ttest).

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Fig. 4. Leptin action on IPSCs synaptic response in POMC neurons from fasted mice. (A) Upper panel showed time-course of bath application of leptin (100nM) on a recorded POMC neuron. Lower panel indicated representative sIPSCs recorded in whole-cell mode. (B) The bar graph represents mean±s.e.m. before and after 10, 20, 30 mins of leptin application (* p < 0.05, paired t-test).

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