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J Physiol 589.3 (2011) pp 639–651 639

Direct and indirect control of orexin/hypocretin neuronsby glycine receptors

Mahesh M. Karnani1, Anne Venner1, Lise T. Jensen2, Lars Fugger3 and Denis Burdakov 1

1Department of Pharmacology, University of Cambridge, Cambridge, UK 2 Aarhus University Hospital, Aarhus, Denmark 3Nuffield Department of Clinical Neurosciences, John Radcliffe Hospital, University of Oxford, Oxford, UK

Non-technical summary Normal wakefulness relies on brain cells called orexin/hypocretinneurons. Activity of these cells stimulates awakening while their loss produces the sleep disordernarcolepsy. By studying what makes orexin/hypocretin cells more or less active, we can thus gaininsights into how the brain switches between different states of consciousness. We describe anew way to turn orexin/hypocretin cells off using a chemical called glycine. We show that glycineshuts down the electrical activity of orexin/hypocretin neurons from the adult brain, but has theopposite effect in the very young brain. Apart from these direct actions on orexin/hypocretin

cells, glycine also enhances the ability of other nerve cells to communicate with orexin/hypocretinneurons. These data shed new light on the basic chemical and physical mechanisms regulatingorexin/hypocretin neurons, which may also be useful in improving therapeutic strategies fordisorders such as insomnia.

Abstract Hypothalamic hypocretin/orexin (hcrt/orx) neurons promote arousal and rewardseeking, while reduction in their activity has been linked to narcolepsy, obesity and depression.However, the mechanisms influencing the activity of hcrt/orx networks in situ are not fully under-stood. Here we show that glycine, a neurotransmitter best known for its actions in the brainstemand spinal cord, elicits dose-dependent postsynaptic Cl− currents in hcrt/orx cells in acute mousebrain slices. This effect was blocked by the glycine receptor (GlyR) antagonist strychnine and

mimicked by the GlyR agonist alanine. Postsynaptic GlyRs on hcrt/orx cells remained functionalduringbothearlypostnatal and adultperiods, and gramicidin-perforated patch-clamp recordingsrevealed that they progressively switch from excitatory to inhibitory during the first two postnatalweeks. The pharmacological profile of the glycine response suggested that developed hcrt/orx neurons contain α/β-heteromeric GlyRs that lack α2-subunits, whereas α2-subunits are presentin early postnatal hcrt/orx neurons. All postsynaptic currents (PSCs) in developed hcrt/orx cellswere blocked by inhibitors of GABA and glutamate receptors, with no evidence of GlyR-mediatedPSCs. However, the frequency but not amplitude of miniature PSCs was reduced by strychnineand increased by glycine in ∼50% of hcrt/orx neurons. Together, these results provide the firstevidence for functional GlyRs in identified hcrt/orx circuits and suggest that the activity of developed hcrt/orx cells is regulated by two GlyR pools: inhibitory extrasynaptic GlyRs locatedon all hcrt/orx cells and excitatory GlyRs located on presynaptic terminals contacting some

hcrt/orx cells.

(Received 24 August 2010; accepted after revision 2 December 2010; first published online 6 December 2010)

Corresponding authors D. Burdakov or M. Karnani: University of Cambridge, Department of Pharmacology, Tennis

Court Road, Cambridge CB2 1PD, UK. Email: [email protected] and [email protected]

Abbreviations ACSF, artificial cerebrospinal fluid; eGFP, enhanced green fluorescent protein; GlyR, glycine receptor;

hcrt/orx, hypocretin/orexin; PSCs, postsynaptic currents.

C 2011 The Authors. Journal compilation

C 2011 The Physiological Society DOI: 10.1113/jphysiol.2010.198457

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640 M. M. Karnani and others J Physiol 589.3

Introduction

Hypothalamic neurons that produce peptide transmittershypocretins/orexins (hereafter referred to as hcrt/orx cells) are vital regulators of states of consciousness andreward-seeking behaviour. Hcrt/orx cells are located inthe lateral hypothalamic area, but project widely to most

of the brain, where they excite target neurons through twospecific G-protein-coupled receptors (de Lecea et al. 1998;Peyron et al. 1998; Sakurai et al. 1998; Sakurai, 2007).The firing of hcrt/orx neurons promotes wakefulness(Adamantidis et al. 2007), and is so critical for sustainedconsciousness that loss of orexin cells causes severenarcolepsy/cataplexy (Thannickal et al. 2000; Hara et al.2001). Hypocretins/orexins also stimulate feeding andreward-seeking behaviour, and destruction of hcrt/orx neurons impairs fasting-induced locomotor activity, andleads to reduced energy expenditure and obesity (Haraet al. 2001; Yamanaka et al. 2003a ; Mieda et al. 2004).Furthermore, overactivity and underactivity of hcrt/orx cells have been recently linked to anxiety and depression,respectively (Boutrel et al. 2005; Suzuki et al. 2005;Brundin et al. 2007; Ito et al. 2008). Exploring differentways of manipulating hcrt/orx cell activity may thus helpdesign better treatment strategies for neurological andpsychiatric disorders.

The most common physiological way of constrainingthe activity of a neural circuit is by activation of GABAAreceptors, which have been reported to be functional inhcrt/orx cells (Li et al. 2002; Yamanaka et al. 2003b ).However, it is unknown how hcrt/orx cells are affectedby other fast transmitters that constrain neural activity,

such as glycine. Although glycine is best known as aninhibitory neurotransmitter in the brainstem and spinalcord (Werman et al. 1968; Gold & Martin, 1983), glycinereceptors (GlyRs) are also found in several higher brainstructures (van den Pol & Gorcs, 1988; Dieudonne, 1995;Rampon et al. 1996; Hussy et al. 1997; Protti et al. 1997;Danober & Pape, 1998; Flint et al. 1998; Chattipakorn& McMahon, 2002; Mangin et al. 2002; Deleuze et al.2005). Both GABAA and GlyRs are anion channels mainly permeable to Cl−. Thus, their activation can producedifferent effects (excitation or inhibition) depending onthe intracellular Cl− concentration, which varies between

different cell types in adult brain (Tozuka et al. 2005; Choiet al. 2008), as well as between different developmentalstages (Ben-Ari et al. 2007). How these factors affecthcrt/orx neurons is unknown, because their responses toglycine have not been examined, whereas their responsesto GABA have only been examined using whole-cellrecordings, where the intracellular Cl− concentration isartificially fixed.

Here, we study the electrical responses of identifiedhcrt/orx neurons to glycine and other known modulatorsof GlyRs. We find that hcrt/orx cells express functional

GlyRs from early postnatal stages through to adulthood.The effect of activation of postsynaptic GlyR Cl− channelsprogressively changes from excitation to inhibition duringthe development of the hcrt/orx network. In addition, indeveloped hcrt/orx circuits, presynaptic GlyRs regulatethe release of both glutamate and GABA onto hcrt/orx cells.

Methods

Preparation of living brain tissue

All animal procedures were performed in accordancewith the Animals (Scientific Procedures) Act 1986 UK,following guidelines in Drummond (2009), and approvedby local animal welfare committees of the University of Cambridge. Transgenic orexin-eGFP mice were used toidentify and study hcrt/orx neurons. These mice expressenhanced green fluorescent protein (eGFP) under the

control of the prepro-orexin promoter, resulting in highly specific targeting of eGFP only to hcrt/orx neurons,as extensively characterized previously (Yamanaka et al.2003a ; Burdakov et al. 2006; Williams et al. 2007, 2008).Mice were maintained on a 12 h light–dark cycle (lightson at 08:00 h) and had free access to food and water.Coronal slices 250 µm thick containing the lateral hypo-thalamus were prepared from mice (ages as indicated inthefigure legends). Mice were killedby cervical dislocationduring the light phase and rapidly decapitated. Brainswere quickly removed and placed into ice-cold ACSF. Ablock of brain tissue was glued to the stage of a CampdenVibroslice for slicing while immersed in ice-cold ACSF.After a 1 h recovery period at 35◦C in ACSF, slices wereused for recordings within∼8 h.

Solutions

ACSF was continuously gassed with 95% O2 and 5% CO2,and contained (in mM): 125 NaCl, 2.5 KCl, 2 MgCl2, 2CaCl2, 1.2 NaH2PO4, 21 NaHCO3 and 1 D-(+)-glucose.For standard whole-cell recordings, three types of intra-cellular (pipette) solutions were used. ‘High-Cl−’ pipettesolution contained (in mM): 130 KCl, 0.1 EGTA, 10

Hepes, 5 K2ATP, 1 NaCl, 2 MgCl2, 40 sucrose, pH 7.3with KOH. ‘Low-Cl−’ pipette solution contained (inmM): 120 potassium gluconate, 10 KCl, 0.1 EGTA, 10Hepes, 5 K2ATP, 1 NaCl, 2 MgCl2, pH 7.3 with KOH.The solution containing 43 mM Cl− used in Fig. 1D contained (in mM): 38 KCl, 92 potassium gluconate, 0.1EGTA, 10 Hepes, 5 K2ATP, 1 NaCl, 2 MgCl2, pH 7.3with KOH. Liquid junction potentials for the low-Cl−

and 43mM Cl− solutions were estimated to be 10.1and 6.0 mV, respectively, and have been subtracted fromthe measurements. For gramicidin-perforated whole-cell


C 2011 The Physiological Society




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J Physiol 589.3 Glycine receptors in brain orexin circuits 641

recordings we filled pipettes with (in mM) 130 KCl, 0.1EGTA, 10 Hepes, 5 K2ATP, 1 NaCl, 2 MgCl2, pH 7.3with KOH and between 300 and 600 µg ml−1 gramicidin(mix of A, B, C and D isoforms, Sigma). A stock solutionof 100mg ml−1 gramicidin was prepared in DMSO with25mgml−1 Pluronic F-127. The hypo-osmotic (‘–30%osmolarity’) stimulation protocol shown in Fig. 5B was

based on Hussy et al. (1997), and consisted of switchingfrom a control solution (ASCF that contained 78 mM NaCland 94 mM sucrose) to a hypo-osmotic solution (same ascontrol solution but without sucrose).

Drugs

The following drugs were added to the extracellularsolution where indicated: 0.05–5 mM glycine (Sigma),50µM (2R )-amino-5-phosphonovaleric acid (AP5),10µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX),

10µM dizocilpine maleate (MK801), 50 or 100µMpicrotoxin (PiTX), 3µM gabazine, 0.001–3µM strychnine,1µM tetrodotoxin (TTX), 100µM cyclothiazide (CTZ)and5mM L-α-alanine.AlldrugswereobtainedfromSigmaor Tocris (UK). Chemicals were applied extracellularly by bath superfusion. All drugs were dissolved in waterexcept PiTX and CTZ, which were dissolved in ethanoland DMSO, respectively (0.1% final concentration).Glutamatergic mPSCs were recorded in the presenceof 3µM gabazine and 1µM TTX, and verified asglutamatergic by blockade with 10µM CNQX. GABAergicmPSCswererecordedinthepresenceof50µM AP5, 10µMCNQX, 10µM MK801 and 1µM TTX, and verified asGABAergic by blockade with 3 µM gabazine.

Recording and analysis

Living orexin-eGFP neurons were visualized in brainslices using an Olympus BX50WI upright microscopeequipped with oblique illumination optics, a mercury lamp and filters for visualizing eGFP-containing cells.Somatic recordings were carried out at 37◦C usingan EPC 10 patch-clamp amplifier controlled by Pulseand Patchmaster software (HEKA Elektronik, Germany).

Patch pipettes were made from borosilicateglass, and theirtip-resistances ranged from 3 to 8 M (3–5 M withhigh-Cl− and 5–8 M with low-Cl− pipette solution).Slices were placed in a submerged-type chamber (volume∼2 ml, solution flow rate 2.5 ml min−1) and anchoredwith a nylon string grid stretched over platinum wire.In standard whole-cell mode, only cells with accessresistances below 20 M were accepted for analysis.In gramicidin-perforated mode, access resistances werebelow 100 M. Signals were low-pass filtered at 3 kHz anddigitized at 7 kHz. Current–voltage (I–V ) relationships

shown inFigs 1D , 2D and5B ,wereobtainedbyperformingvoltage-clamp ramps from −10 to −140 mV at a rate of 0.1 mVms−1.

To studyevokedpostsynaptic currents (Fig. 5A ,right),aconcentric bipolar stimulation electrode (World PrecisionInstruments) was placed within the lateral hypothalamus50–200µm away from the recorded cell. Stimulatory

pulse characteristics (100–200µA, 0.2 ms, 0.2 Hz) werecontrolled by a DS3 isolated stimulator (Digitimer, UK).Theseresponseswereconfirmedtobesynapticbyblockadewith a cocktail of ionotropic glutamate, GABAA andglycine receptor blockers at the end of each experiment.During our analysis of mEPSCs, we looked at kineticsof the individual synaptic events (using Minianalysis,Synaptosoft, Fort Lee, NJ, USA), and confirmed that thetime constant of decay was 0.2,F test).






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Results

GlyR modulators regulate postsynaptic Cl− currents

in hcrt/orx neurons

To explorethe effects of glycine on identifiedhcrt/orx cells,we first analysedthe effect of glycine on membrane current

using standard whole-cell voltage-clamp recordings fromidentified hcrt/orx cells in acutely isolated mouse brainslices. Glycine (0.5 mM) elicited large membrane currentsin all (>50) cells tested (Fig. 1A ). The amplitudes of glycine-induced currents were not significantly affectedby pharmacological synaptic isolation (Fig. 1A and B ;

Figure 1. Biophysical properties of postsynaptic GlyRs in hcrt/orx neuronsData in this figure are from P13–27 mice. A, typical current response to 0.5 mM glycine with the high Cl−

intracellular solution (holding potential = −60 mV). Current was 790.4 ± 91.5 pA, n = 7. All cells responded in

this way (n = 50/50). B, typical current response to 0.5 mM glycine during blockade of fast glutamatergic or

GABAergic neurotransmission and action potentials (holding potential = −60 mV). Current was 519.6 ± 94.4 pA,

n = 4, P > 0.05 by unpaired t test compared to the control data shown in A (which was collected from a different

set of cells). C , same recording conditions as in A and B (but a different set of cells). Left, 3 µM strychnine

completely blocked the response to 0.5 mM glycine (n = 5, see control trace shown in A for comparison). Right,

dose–response of strychnine-induced inhibition of the current produced by 1 mM glycine (n> 3 cells per point,

IC50 = 0.22 µM). D, net current–voltage relationships of conductance activated by 0.5 mM glycine with intracellular

solutions containing 15 mM (n = 5) and 43 mM Cl (n = 6), and in the presence of 3 µM strychnine (n = 5). Values

are means (black) and S.E.M. (grey). E , dependence of the reversal potential of current activated by 0.5 m M glycine

on intracellular [Cl]; dashed line represents theoretical (Nernstian) Cl− reversal potential, n = 5 for each point.






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Figure 2. Pharmacological properties of postsynaptic GlyRs in

hcrt/orx neurons

control, 790.4± 91.5 pA at –60 mV, n = 7 cells; synapticblockers, 519.6± 94.4 pA at −60 mV, n = 4 cells,P > 0.05 by unpaired t test), but were dose-dependently blocked by strychnine, a selective antagonist of GlyRs(Fig. 1C , n = 25). The current–voltage relationship of theglycine-activated current exhibited outward rectification(Fig. 1D , n = 11), in agreement with known biophysical

properties of GlyR Cl− channels (Rajendra et al. 1997). Asthe intracellular chloride concentration was progressively reduced, the reversal potential of the glycine-activatedcurrent became progressively more negative, in goodagreement with the Nernst prediction for a Cl−-selectiveion channel (Fig. 1D and E ). Together, these datastrongly imply that hcrt/orx cells express functionalstrychnine-sensitive GlyRs.

To functionally characterize the type of GlyR expressedby hcrt/orx neurons, we examined the dose–responserelationship of glycine activation of membrane currents.Bath application of 50µM t o 5 mM glycine induced

dose-dependent responses with EC50 of 0.7 mM (Fig. 2A ).Application of 100µM picrotoxin, which is expectedto block α-homomeric GlyRs but not α/β-heteromericGlyRs (Pribilla et al. 1992), did not reduce the amplitudeof glycine-induced currents (Fig. 2B ; at −60 mV,picrotoxin= 620.0± 19.9 pA; control= 673.6± 25.4 pA,n = 5, P > 0.1), suggesting that heteromeric GlyRsare involved. Application of 100µM cyclothiazide,a selective blocker of α2-containing GlyRs (Zhanget al. 2008b ), also did not affect the amplitude of glycine-induced currents (Fig. 2B ; at −60 mV, cyclo-thiazide= 746.8± 101.5 pA; control 803.0± 179.4 pA,n = 4, P > 0.5), arguing against the presence of α2 GlyRsin developed hcrt/orx cells. Like glycine, L-α-alanine,another GlyR agonist (Rajendra et al. 1997), elicitedstrychnine-sensitive, Cl−-selective, outwardly rectifyingmembranecurrents (Fig. 2C ; at−60 mV, 721.9± 75.4 pA,n = 22; Fig. 2D , reversal potential, −63.3± 3.7 mV,predicted E Cl =−58.5 mV, n = 5).

Data in this figure are from P13–27 mice. A, dose–response curve for

glycine currents recorded with the high Cl− intracellular solution at

−60 mV (n > 3 for each point), EC50 = 0.7 mM. B, left, response to

0.5 mM glycine is not blocked by 0.1 mM picrotoxin (PiTX)

(620.0± 19.9 pA, n = 5, not significantly different from control,

673.6± 25.4 pA, P > 0.1). Right, response to 0.5 mM glycine is not

blocked by 0.1 mM cyclothiazide (CTZ) (746.8 ± 101.5 pA, n = 4,

not significantly different from control, 803.0 ± 179.4 pA, P > 0.5).

Holding potential was −60 mV. C , 5 mM alanine induces an inward

current (721.9 ± 75.4 pA, n = 22, holding potential is −60 mV, high

Cl− intracellular solution). D, Current-voltage relationship of current

induced by 5 mM alanine in the absence (n = 5), and presence of

strychnine (1 µM, n = 3). The reversal potential was −63.3 ± 3.7 mV

in 15 mM intracellular Cl− (predicted Nernst E Cl = −58.8 mV). Values

are means (black) and S.E.M. (grey).






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Figure 3. Properties of glycine-induced currents in neonatal

(P3–5) hcrt/orx cells

Responses of hcrt/orx neurons to glycine change

during development

The above experiments used postnatal day (P) 13–27mice. To examine developmental properties of hcrt/orx cell GlyRs, we also studied neonatal (P3–5) mice. Theneonatal hcrt/orx cells also displayed dose-dependent

glycine currents (Fig. 3A ), and tended to be slightly moresensitive to glycine (EC50 = 0.44 mM) than mature cells(EC50 = 0.7mM; see Fig. 2A ), although the difference insensitivity did not reach statistical significance (P > 0.3,F test). Application of 100µM picrotoxin or 100µMcyclothiazide significantly reduced the amplitude of glycine-induced currents in P3–5 hcrt/orx cells (Fig. 3B and C , statistics for rawdata andnormalized responses aregivenin thefigure legend).This suggests that, in contrasttoP13–27 hcrt/orx cells where picrotoxin and cyclothiazidewere ineffective (Fig. 2B ), neonatal hcrt/orx cells containsome α-homomeric GlyRs (as implied by picrotoxinsensitivity) andα2-containing GlyRs (as implied by cyclo-thiazide sensitivity).

To determine the physiological effect (depolarizationvs. hyperpolarization) of glycine-induced currents inhcrt/orx cells, we monitored the membrane potentialusing gramicidin-perforated patch recordings, whichpreserve the endogenous Cl− concentration in hcrt/orx cell cytosol, and so allow GlyR Cl− channels to exert theirtrue physiological effects on the membrane potential. Ingramicidin-perforated patch recordings, glycine elicitedrobust hyperpolarization in 100% of P19 hcrt/orx cells,but as we examined progressively younger animals,we observed an increasing proportion of depolarizing

responses (Fig. 4A and C , at least 4 cells were analysedat each time point). We confirmed that these differences(hyperpolarizing vs . depolarizing) were not due todifferences in resting membrane potentials (RMP) inglycine-hyperpolarized and glycine-depolarized cells, andthus probably resulted from developmental changesin the transmembrane Cl− gradient (RMP of hyper-polarized cells was −47.0± 1.0 mV, RMP of depolarizedcells was −45.8± 1.6 mV, n = 22 and 12, respectively,P > 0.3). We also carried out control experiments in the

A, dose–response curve for glycine currents recorded with the high

Cl− intracellular solution at −60 mV (n> 3 for each point),

EC50 = 0.44 mM. B, top, response to 0.5 mM glycine is reduced by

0.1 mM PiTX (control = 1163.5± 178.3 pA,

PiTX = 624.3± 62.5 pA, n = 4, P < 0.05). Bottom, response to

0.5 mM glycine is reduced by 0.1 mM cyclothiazide (CTZ)

(control= 950.6± 171.6 pA, CTZ = 771.1± 165.8 pA, n = 4,

P < 0.05). Holding potential was −60 mV. C , comparison of

picrotoxin and cyclothiazide sensitivity in P3–5 and P14–20 cells

(n = 4 to 5 cells in each group). The responses were normalized to

the responses without the drugs measured in the same cell. ns, not

significant (P > 0.1), ∗∗∗P < 0.005, ∗∗P < 0.02.






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presence of the NMDA receptor blocker AP5 (50 µM),which confirmed that glycine action on the NMDAreceptor (Dingledine et al. 1999) did not contribute toour results (Fig. 4B , depolarization by 0.5 mM glycinewithout AP5= 27.8± 2.1 mV; depolarization by 0.5 mMglycine with AP5= 29.3± 2.7 mV; n = 3; P > 0.2). Ouranalysis of cells from P3 to P19 indicated that the

switch from depolarizing to hyperpolarizing responsesto glycine is completed between P15 and P19 (Fig. 4C ).These results probably reflect a gradual developmentof the transmembrane Cl− gradient in hcrt/orx cellsduring the first weeks of life (see Discussion). Finally, wealso confirmed that strychnine-sensitive glycine currentswere present in old (P140) mice (current induced by 0.5 mM glycine without strychine= 43.7± 5.6pA pF−1;with 3µM strychnine= 0.2± 0.1pA pF−1; P < 0.001,n = 4, holding potential=−60 mV, data not shown),indicating that hcrt/orx cells can be controlled by GlyRsthroughout the animal’s lifetime.

Presynaptic GlyRs regulate glutamate and GABA

release onto hcrt/orx neurons

When excitatory spontaneous post-synaptic currents(sPSCs) were blocked by AP5, CNQX and MK801, theremaining sPSCs had a frequency of 1.01± 0.19 Hz(Fig. 5A , n = 5 cells). However, these sPSCs wereexclusively GABAergic, with no contributions from post-synaptic GlyRs, because they were completely abolishedby 3µM gabazine (Fig. 5A ), which blocks GABAergiccurrents without affecting glycinergic currents (Chery & de Koninck, 1999; Mori et al. 2002; Beato, 2008).This suggests that all spontaneous synaptic currentsobserved in hcrt/orx cells are mediated by glutamateand GABA receptors. We also analysed the amplitudeof evoked PSCs (ePSCs, see Methods), and found itunaffected by strychnine (Fig. 5A ; ePSC amplitude instrychnine was 100.3± 8.6% of control, n = 4, P > 0.5).This confirms that postsynaptic GlyRs on hcrt/orx cellsare not activated by synaptic release (see Discussion).Apart from synaptically released glycine, GlyRs could alsobe activated by taurine released from astrocytes underhypo-osmotic conditions (Hussy et al. 2000). However, we

found that membrane potential and membrane currentresponses of hcrt/orx neurons to a hypo-osmotic stimulus(30% reduction in osmolarity, see Methods) did not havea strychnine-sensitive component (Fig. 5B , membranepotential data: control response= 19.7± 1.3 mVdepolarization; strychnine response= 21.4± 2.9 mVdepolarization; P > 0.5; membrane current data: at−60mV, control=−105.4± 46.5 pA, strychnine=−105.2± 36.6 pA, n = 3, P > 0.5).

To test if, in addition to the postsynaptic effectsdescribed in theprevious sections,GlyRs may also regulate

Figure 4. Developmental profile of glycine effects on hcrt/orx

neurons

Ages of mice are indicated near corresponding traces and diagram.

A, representative depolarizing (top) and hyperpolarizing (bottom)

perforated-patch current-clamp recordings of glycine (0.5 mM)

responses in hcrt/orx neurons. These experiments were

performed at zero holding current. B, representative recording

showing that the amplitude of glycine response is not affected bythe presence of AP5 (depolarization by 0.5 mM glycine without AP5,

27.8 ± 2.1 mV; depolarization by 0.5 mM glycine with AP5,

29.3 ± 2.7 mV; n = 3; P > 0.2). This set of experiments was

performed using whole-cell configuration with high Cl− intracellular

solution at zero holding current. C , summary of responses in A at

different ages; responses were categorized as depolarizing or

hyperpolarizing and summarized as a bar graph of at least 4 cells at

each time point.






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Figure 5. Postsynaptic GlyRs are not activated

by synaptic release or hypo-osmolarity, but

presynaptic GlyRs tonically enhance GABA

release onto some hcrt/orx neurons

A, data in this panel are from P13–27 mice. Left

(top), an example of voltage-clamp recording in thepresence of AP5, CNQX and MK801, showing

remaining PSCs (mean frequency 1.01 ± 0.19 Hz,

n = 5 cells; high Cl− intracellular solution,

voltage-clamp at −60 mV). Left (bottom), all

remaining PSCs are abolished by 3 µM gabazine

(mean frequency 0.00 ± 0.00 Hz, n = 11 cells).

Right, an example of evoked PSCs (each trace is

mean of 10 responses) in the absence (grey trace)

and presence (black trace) of 1 µM strychnine

(amplitude in strychnine was 100.3 ± 8.6% of

control, n = 4 cells, P > 0.5). B, data in this panel

are from P13–27 mice. Left, membrane potential

responses to hypo-osmolarity (see Methods) in the

absence (top) and presence (bottom) of 3 µM

strychnine (control response, 19.7 ± 1.3 mV;strychnine response, 21.4 ± 2.9 mV; P > 0.5).

Right, net currents (obtained using voltage ramps,

see Methods) induced by hypo-osmolarity in the

absence (cntrl) and presence (stry) of 3 µM

strychnine (at −60 mV: control, −105.4± 46.5 pA;

strychnine,−105.2± 36.6 pA, n = 3, P > 0.5). C ,

data in this panel are from P22–27 mice. Examples

of GABA mPSCs in the presence and absence of

1 µM strychnine (recorded with 50 µM AP5, 10 µM

CNQX, 10 µM MK801 and 1 µM TTX in bath,−60 mV holding potential, high-Cl− pipette

solution). D, data in this panel are from P22–27

mice. Left, inter-event intervals (IEI) of GABA mPSCs

from 4 out of 9 cells that responded to 1 µM

strychnine (control, continuous line; strychnine,dashed line). Kolmogorov–Smirnov test indicated

significant difference between the two conditions,

P < 0.01. Inset shows means of the 4/9 cells in

which strychnine increased IEI (27.6 ± 8.9%

increase relative to control, ∗P < 0.02). Right,

amplitudes of the mPSCs from the left-hand panel

(control, continuous line; strychnine, dashed line),

P > 0.05 by Kolmogorov–Smirnov test; inset shows

means (strychnine decreased amplitude by

1.1 ± 4.0% relative to control, P > 0.8).






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glutamate and GABA terminals contacting hcrt/orx cells,we blocked action potential-mediated synaptic releasewith tetrodotoxin, and examined the frequency of theresulting miniature post-synaptic currents (mPSCs).We found that strychnine significantly decreased thefrequency, but did not affect the amplitude, of GABAergicmPSCs in 4 out of 9 cells tested (Fig. 5C and D , statistics

are given in legend to Fig. 5D ), suggesting that pre-synaptic GlyRs tonically enhance the release of GABAonto hcrt/orx cells. Surprisingly, we also observed thatstrychnine had a significant inhibitory effect on thefrequency (but not amplitude) of glutamatergic mPSCs in4 out of9 cellstested (Fig. 6, statistics are given in legend to(Fig. 6B ).

We also examined the modulation of mPSCs by glycine. In these experiments, we had to voltage-clampthe postsynaptic cells near the reversal potential of GlyR-mediated responses, because we found that atother holding potentials, the large postsynaptic channel

noise induced by glycine (Fig. 1A ) obscured the mPSCs.It was thus only possible to examine the effects onglutamate mPSCs, because GABA mPSCs reverse atthe same potential as glycine currents and thus areinvisible with this voltage-clamp protocol (−60mVholding potential, low-Cl− pipette solution). We foundthat glycine significantly increased the frequency (butnot amplitude) of glutamate mPSCs in 4 out of 7 cellstested (Fig. 7, statistics are given in legend to Fig. 7B ),providing further evidence for the existence of apresynaptic population of GlyRs.

Discussion

Although the importance of GlyRs in brainstem andspinal cord is well established (Rajendra et al. 1997),the function of GlyRs in higher brain areas is lessunderstood. Despite previous reports of expression of GlyRs in the hypothalamus (van den Pol & Gorcs, 1988;Rampon et al. 1996), their role in shaping the activity of neurochemically and functionally defined hypothalamicneurons remained largely unknown. This is the first reportlinking modulation of glycine receptors to the activity of identified hcrt/orx cells, key hypothalamic players in

the regulation of wakefulness, energy expenditure andreward seeking. Our results provide evidence that theactivity of hcrt/orx cells is regulated by functional GlyRslocated on both postsynaptic sites on the hcrt/orx cellmembrane and on glutamatergic and GABAergic synapticterminals contacting hcrt/orx cells. Since the action of glycine on GlyRs on mature hcrt/orx cells was hyper-polarizing, whereas the action on presynaptic GlyRsincreased synaptic release onto hcrt/orx neurons, thesedata reveal two distinct mechanisms for modulating thefiring of hcrt/orx neurons.

We found that GlyR agonists triggered large post-synaptic Cl− currents in hcrt/orx cells. This responsewas directly mediated by GlyRs on the recordedneuron, as shown by lack of blockade by TTX andblockers of ionotropic GABA and glutamate receptors,

Figure 6. Presynaptic GlyRs tonically enhance glutamate

release onto hcrt/orx neurons

Data in this figure are from P22–27 mice. A, examples of glutamatemPSCs in the presence and absence of 1 µM strychnine (recorded

with 3 µM gabazine and 1 µM TTX in bath, −60 mV holding

potential, low-Cl− pipette solution). B, top, inter-event intervals (IEI)

of glutamate mPSCs from 4 out of 9 cells that responded to 1 µM

strychnine (control, continuous line; strychnine, dashed line).

Kolmogorov-Smirnov test indicated significant difference between

the two conditions, P < 0.0001. Inset shows means of the 4/9 cells

in which strychnine increased IEI (28.1 ± 3.5% increase relative to

control, ∗∗∗P < 0.0001). Bottom, amplitudes of the mPSCs from the

top panel (control, continuous line; strychnine, dashed line), P > 0.4

by Kolmogorov–Smirnov test; inset shows means (strychnine

increased amplitude by 3.2 ± 2.2% relative to control, P > 0.1).






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Figure 7. Glycine enhances glutamate release onto hcrt/orx

neurons

Data in this panel are from P19–27 mice. A, examples of glutamate

mPSCs in the presence and absence of 0.5 mM glycine. Recording

performed at −60 mV, low-Cl

−

pipette solution. B, top, inter-eventintervals (IEI) of glutamate mPSCs from 4/7 cells that responded to

0.5 mM glycine (control, continuous line; strychnine, dashed line).

Kolmogorov–Smirnov test indicated significant difference between

the two conditions, P < 0.0001. Inset shows means of the 4/7 cells

in which glycine decreased IEI (29.2 ± 1.9% decrease relative to

control, ∗∗∗P < 0.001). Bottom, amplitudes of the mPSCs from the

top panel (control, continuous line; glycine, dashed line), P > 0.3 by

Kolmogorov–Smirnov test; inset shows means (glycine decreased

amplitude by 1.4 ± 1.7% relative to control, P > 0.2).

and by blockade by strychnine. Our comparison of agonist and antagonist potencies suggested that hcrt/orx cell GlyRs undergo a change in subunit compositionduring development. Neonatal hcrt/orx cells appeared tocontain a significant proportion of α-homomeric andα2-containing GlyRs, as implied by cyclothiazide andpicrotoxin sensitivity of glycine responses. In contrast, the

glycine responses of mature hcrt/orx cells were insensitiveto cyclothiazide and picrotoxin, suggesting that they contain heteromeric GlyR channels that lack α2 subunits.This is consistent with other studies (e.g. Malosio et al.1991) suggesting that the expression of α2 subunitsdecreases after birth. Although our glycine EC50 valuescould be overestimates due to bath application in a slice,the trend toward greater sensitivity in neonatal hcrt/orx is in line with a postnatal shift from α2-containing GlyRs(EC50 ≈ 300µM, Grenningloh et al. 1990; Schmieden et al.1992), toward α3-containing GlyRs (EC50 ≈ 0.75 mM,Kuhse et al. 1990), rather than α1-containing GlyRs of

∼200µM, (Lewis et al. 1998).Although in some neurocircuits GlyRs are

developmentally down- or up-regulated during thefirst few postnatal weeks (Malosio et al. 1991; Turecek & Trussell, 2002; Kubota et al. 2010) and seem to havedevelopmental roles (Flint et al. 1998), our data indicatethat functional postsynaptic GlyRs are present on orexinneurons well into adulthood. While this does not precludea developmental role for the GlyRs, it does imply that they serve some function in the adult. Gramicidin-perforatedpatch-clamp recordings revealed that when [Cl−]i wasunperturbed, most orexin neurons hyperpolarized inresponse to glycine application after P15, whereas betweenP3 and P10 around half of the cells were depolarized by glycine. The time-line of Cl− gradient maturation, whichis thought to result from developmental up-regulationof plasmalemmal K+/Cl− cotransporter KCC2 (Riveraet al. 1999), was similar to that previously reported inthe hypothalamus (Gao & van den Pol, 2001; Wanget al. 2001) and in other brain regions (Ben-Ari et al.2007).

We did not observe any endogenous glycine-mediatedsynaptic currents in hcrt/orx cell membrane, suggestingthat synapses contacting hcrt/orx cells do not releaseglycine, and modulators of the postsynaptic GlyRs in

hcrt/orx cells are likely to come from extrasynapticsources. For example, the levels of glycine and alaninein the extracellular space change during fasting andfeeding. During prolonged fasting in man, the plasmaconcentration of alanine falls significantly within 3 dayswhereas glycine rises within 10 days (Adibi, 1968; Feliget al. 1969). Interestingly, Adibi (1968) also showed thatduring isocaloric protein-free dieting, plasma alanine isrobustly elevated. As changes in intragastric levels of amino acids subsequently lead to robust changes in aminoacid levels in the lateral hypothalamus (Choi et al. 1999),






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it is possible that under certain circumstances glycine(and/or alanine) may tonically regulate hcrt/orx neuronsvia extrasynaptically located GlyRs, as proposed for someother central neurons (Flint et al. 1998; Mori et al.2002; Meier et al. 2005; Zhang et al. 2008a ). Anotherendogenous ligand of GlyRs is taurine, which in somehypothalamic regions may be released from astrocytes

under hypo-osmotic conditions (Hussy et al. 2001).However, we found that responses of hcrt/orx neuronsto hypo-osmolarity did not have a strychnine-sensitivecomponent, arguing against this mechanism in hcrt/orx cells.

In terms of effects of GlyR modulation on endogenoussynaptic release of other transmitters onto hcrt/orx cells,we found that, in about 50% of hcrt/orx cells, GlyR inhibition reduced the frequency of both GABA andglutamate mPSCs, while GlyR activation increased thefrequency of glutamate mPSCs (we could not measureeffects of glycine on GABA mPSCs for technical reasons

described above). Because mPSC frequency is a standardmeasure of presynaptic release probability, the simplestexplanation for these results is that there are excitatory GlyRs located on glutamate and GABA synaptic terminalscontacting hcrt/orx cells, which are tonically active inour brain slice preparation. We note that, althoughthe classical postsynaptic action of glycine is hyper-polarizing and inhibitory, there is evidence from otherpreparations that GlyRs located on presynaptic nerveterminals can instead evoke depolarizing Cl− currents,enhancing transmitter release by increasing the activity of depolarization-activated Ca2+ channels (Turecek &Trussell, 2001; Jeong et al. 2003; Ye et al. 2004; Leeet al. 2009). Our data suggest that similar excitatory GlyRs operate in both GABA and glutamate terminalscontacting hcrt/orx neurons. Presumably the synapsescontaining presynaptic GlyRs comprise only a smallfraction of total synapse number on hcrt/orx cells, sincewe could not resolve a significant effect of strychnineon responses involving synchronized activation of many synapses (Fig. 5A , right trace). It remains to be determinedwhether presynaptic GlyRs on the glutamate and GABAterminals are activated separately or together, and wecan only speculate about the physiological source(s) of activators of the presynaptic GlyRs on hcrt/orx cells.

For example, evidence dating back to the early 1980spoints to an existence of an inhibitory glycinergiccorticohypothalamic pathway (Kita & Oomura, 1981,1982).

In summary, the functional GlyRs in hcrt/orx networksrepresent a previously undescribed way to control theactivity of the hcrt/orx system, and could potentially beengaged by GlyR-modulating drugs (Nguyen et al. 2009),and as yet undefined physiological modulators, to controlbrain function.

References

Adamantidis AR, Zhang F, Aravanis AM, Deisseroth K & deLecea L (2007). Neural substrates of awakening probed withoptogenetic control of hypocretin neurons. Nature 450,420–424.

Adibi SA (1968). Influence of dietary deprivations on plasmaconcentration of free amino acids of man. J Appl Physiol 25,52–57.

Beato M (2008). The time course of transmitter at glycinergicsynapses onto motoneurons. J Neurosci 28, 7412–7425.

Ben-Ari Y, Gaiarsa JL, Tyzio R & Khazipov R (2007). GABA: apioneer transmitter that excites immature neurons andgenerates primitive oscillations. Physiol Rev 87,1215–1284.

Boutrel B, Kenny PJ, Specio SE, Martin-Fardon R, Markou A,Koob GF & de Lecea L (2005). Role for hypocretin inmediating stress-induced reinstatement of cocaine-seekingbehavior. Proc Natl Acad Sci U S A 102, 19168–19173.

Brundin L, Bjorkqvist M, Petersen A & Traskman-Bendz L(2007). Reduced orexin levels in the cerebrospinal fluid of

suicidal patients with major depressive disorder. Eur Neuropsychopharmacol 17, 573–579.

Burdakov D, Jensen LT, Alexopoulos H, Williams RH, FearonIM, O’Kelly I et al . (2006). Tandem-pore K+ channelsmediate inhibition of orexin neurons by glucose. Neuron 50,711–722.

Chattipakorn SC & McMahon LL (2002). Pharmacologicalcharacterization of glycine-gated chloride currents recordedin rat hippocampal slices. J Neurophysiol 87, 1515–1525.

Chery N & de Koninck Y (1999). Junctional versusextrajunctional glycine and GABAA receptor-mediatedIPSCs in identified lamina I neurons of the adult rat spinalcord. J Neurosci 19, 7342–7355.

Choi HJ, Lee CJ, Schroeder A, Kim YS, Jung SH, Kim JS et al .

(2008). Excitatory actions of GABA in the suprachiasmaticnucleus. J Neurosci 28, 5450–5459.

Choi YH, Chang N & Anderson GH (1999). An intragastricamino acid mixture influences extracellular amino acidprofiles in the lateral hypothalamic area of freely movingrats. Can J Physiol Pharmacol 77, 827–834.

Danober L & Pape HC (1998). Strychnine-sensitive glycineresponses in neurons of the lateral amygdala: anelectrophysiological and immunocytochemicalcharacterization. Neuroscience 85, 427–441.

de Lecea L, Kilduff TS, Peyron C, Gao X, Foye PE, Danielson PEet al . (1998). The hypocretins: hypothalamus-specificpeptides with neuroexcitatory activity. Proc Natl Acad Sci

U S A 95, 322–327.Deleuze C, Alonso G, Lefevre IA, Duvoid-Guillou A & Hussy N(2005). Extrasynaptic localization of glycine receptors in therat supraoptic nucleus: further evidence for theirinvolvement in glia-to-neuron communication. Neuroscience 133, 175–183.

Dieudonne S (1995). Glycinergic synaptic currents in Golgicells of the rat cerebellum. Proc Natl Acad Sci U S A 92,1441–1445.

Dingledine R, Borges K, Bowie D & Traynelis SF (1999). Theglutamate receptor ion channels. Pharmacol Rev 51, 7–61.






12/13


Drummond GB (2009). Reporting ethical matters in The Journal of Physiology : standards and advice. J Physiol 587,713–719.

Felig P, Owen OE, Wahren J & Cahill GF Jr (1969). Amino acidmetabolism during prolonged starvation. J Clin Invest 48,584–594.

Flint AC, Liu X & Kriegstein AR (1998). Nonsynaptic glycine

receptor activation during early neocortical development.Neuron 20, 43–53.Gao XB & Van Den Pol AN (2001). GABA, not glutamate, a

primary transmitter driving action potentials in developinghypothalamic neurons. J Neurophysiol 85, 425–434.

Gold MR & Martin AR (1983). Characteristics of inhibitory post-synaptic currents in brain-stem neurones of thelamprey. J Physiol 342, 85–98.

Grenningloh G, Schmieden V, Schofield PR, Seeburg PH,Siddique T, Mohandas TK, Becker CM & Betz H (1990).Alpha subunit variants of the human glycine receptor:primary structures, functional expression and chromosomallocalization of the corresponding genes. EMBO J 9, 771–776.

Hara J, Beuckmann CT, Nambu T, Willie JT, Chemelli RM,

Sinton CM et al . (2001). Genetic ablation of orexin neuronsin mice results in narcolepsy, hypophagia, and obesity.Neuron 30, 345–354.

Hussy N, Bres V, Rochette M, Duvoid A, Alonso G, DayanithiG & Moos FC (2001). Osmoregulation of vasopressinsecretion via activation of neurohypophysial nerveterminals glycine receptors by glial taurine. J Neurosci 21,7110–7116.

Hussy N, Deleuze C, Desarmenien MG & Moos FC (2000).Osmotic regulation of neuronal activity: a new role fortaurine and glial cells in a hypothalamic neuroendocrinestructure. Prog Neurobiol 62, 113–134.

Hussy N, Deleuze C, Pantaloni A, Desarmenien MG & Moos F(1997). Agonist action of taurine on glycine receptors in ratsupraoptic magnocellular neurones: possible role inosmoregulation. J Physiol 502, 609–621.

Ito N, Yabe T, Gamo Y, Nagai T, Oikawa T, Yamada H &Hanawa T (2008). i.c.v. administration of orexin-A inducesan antidepressive-like effect through hippocampal cellproliferation. Neuroscience 157, 720–732.

Jeong HJ, Jang IS, Moorhouse AJ & Akaike N (2003).Activation of presynaptic glycine receptors facilitates glycinerelease from presynaptic terminals synapsing onto rat spinalsacral dorsal commissural nucleus neurons. J Physiol 550,373–383.

Kita H & Oomura Y (1981). Reciprocal connections betweenthe lateral hypothalamus and the frontal complex in the rat:

electrophysiological and anatomical observations. Brain Res 213, 1–16.

Kita H & Oomura Y (1982). Evidence for a glycinergiccortico-lateral hypothalamic inhibitory pathway in the rat.Brain Res 235, 131–136.

Kubota H, Alle H, Betz H & Geiger JR (2010). Presynapticglycine receptors on hippocampal mossy fibers. BiochemBiophys Res Commun 393, 587–591.

Kuhse J, Schmieden V & Betz H (1990). Identification andfunctional expression of a novel ligand binding subunit of theinhibitory glycine receptor. J Biol Chem 265, 22317–22320.

Lee EA, Cho JH, Choi IS, Nakamura M, Park HM, Lee JJ et al .(2009). Presynaptic glycine receptors facilitate spontaneousglutamate release onto hilar neurons in the rat hippocampus.

J Neurochem 109, 275–286.Lewis TM, Sivilotti LG, Colquhoun D, Gardiner RM, Schoepfer

R & Rees M (1998). Properties of human glycine receptorscontaining the hyperekplexia mutation α1(K276E),

expressed in Xenopus oocytes. J Physiol 507, 25–40.Li Y, Gao XB, Sakurai T & Van Den Pol AN (2002).Hypocretin/orexin excites hypocretin neurons via a localglutamate neuron-A potential mechanism for orchestratingthe hypothalamic arousal system. Neuron 36, 1169–1181.

Malosio ML, Marqueze-Pouey B, Kuhse J & Betz H (1991).Widespread expression of glycine receptor subunit mRNAsin the adult and developing rat brain. EMBO J 10,2401–2409.

Mangin JM, Guyon A, Eugene D, Paupardin-Tritsch D &Legendre P (2002). Functional glycine receptor maturationin the absence of glycinergic input in dopaminergic neuronesof the rat substantia nigra. J Physiol 542, 685–697.

Meier JC, Henneberger C, Melnick I, Racca C, Harvey RJ,

Heinemann U et al . (2005). RNA editing produces glycinereceptor α3(P185L), resulting in high agonist potency. Nat Neurosci 8, 736–744.

Mieda M, Williams SC, Sinton CM, Richardson JA, Sakurai T &Yanagisawa M (2004). Orexin neurons function in anefferent pathway of a food-entrainable circadian oscillator ineliciting food-anticipatory activity and wakefulness. J Neurosci 24, 10493–10501.

Mori M, Gahwiler BH & Gerber U (2002). β-Alanine andtaurine as endogenous agonists at glycine receptors in rathippocampus in vitro . J Physiol 539, 191–200.

Nguyen HT, Li KY, daGraca RL, Delphin E, Xiong M & Ye JH(2009). Behavior and cellular evidence for propofol-inducedhypnosis involving brain glycine receptors. Anesthesiology 110, 326–332.

Peyron C, Tighe DK, Van Den Pol AN, de Lecea L, Heller HC,Sutcliffe JG & Kilduff TS (1998). Neurons containinghypocretin (orexin) project to multiple neuronal systems. J Neurosci 18, 9996–10015.

Pribilla I, Takagi T, Langosch D, Bormann J & Betz H (1992).The atypical M2 segment of the β subunit conferspicrotoxinin resistance to inhibitory glycine receptorchannels. EMBO J 11, 4305–4311.

Protti DA, Gerschenfeld HM & Llano I (1997). GABAergic andglycinergic IPSCs in ganglion cells of rat retinal slices. J Neurosci 17, 6075–6085.

Rajendra S, Lynch JW & Schofield PR (1997). The glycine

receptor. Pharmacol Ther 73, 121–146.Rampon C, Luppi PH, Fort P, Peyron C & Jouvet M (1996).

Distribution of glycine-immunoreactive cell bodies andfibers in the rat brain. Neuroscience 75, 737–755.

Rivera C, Voipio J, Payne JA, Ruusuvuori E, Lahtinen H, LamsaK et al . (1999). The K+/Cl− co-transporter KCC2 rendersGABA hyperpolarizing during neuronal maturation. Nature 397, 251–255.

Sakurai T (2007). The neural circuit of orexin (hypocretin):maintaining sleep and wakefulness. Nat Rev Neurosci 8,171–181.






13/13


Sakurai T, Amemiya A, Ishii M, Matsuzaki I, Chemelli RM,Tanaka H et al . (1998). Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupledreceptors that regulate feeding behavior. Cell 92,573–585.

Schmieden V, Kuhse J & Betz H (1992). Agonist pharmacology of neonatal and adult glycine receptor alpha subunits:

identification of amino acid residues involved in taurineactivation. EMBO J 11, 2025–2032.Suzuki M, Beuckmann CT, Shikata K, Ogura H & Sawai T

(2005). Orexin-A (hypocretin-1) is possibly involved ingeneration of anxiety-like behavior. Brain Res 1044,116–121.

Thannickal TC, Moore RY, Nienhuis R, Ramanathan L, GulyaniS, Aldrich M et al . (2000). Reduced number of hypocretinneurons in human narcolepsy. Neuron 27, 469–474.

Tozuka Y, Fukuda S, Namba T, Seki T & Hisatsune T (2005).GABAergic excitation promotes neuronal differentiation inadult hippocampal progenitor cells. Neuron 47, 803–815.

Turecek R & Trussell LO (2001). Presynaptic glycine receptorsenhance transmitter release at a mammalian central synapse.

Nature 411, 587–590.Turecek R & Trussell LO (2002). Reciprocal developmental

regulation of presynaptic ionotropic receptors. Proc Natl Acad Sci U S A 99, 13884–13889.

Van Den Pol AN & Gorcs T (1988). Glycine and glycinereceptor immunoreactivity in brain and spinal cord. J Neurosci 8, 472–492.

Wang YF, Gao XB & Van Den Pol AN (2001). Membraneproperties underlying patterns of GABA-dependent actionpotentials in developing mouse hypothalamic neurons. J Neurophysiol 86, 1252–1265.

Werman R, Davidoff RA & Aprison MH (1968). Inhibitory of glycine on spinal neurons in the cat. J Neurophysiol 31,81–95.

Williams RH, Alexopoulos H, Jensen LT, Fugger L & Burdakov D (2008). Adaptive sugar sensors in hypothalamic feedingcircuits. Proc Natl Acad Sci U S A 105, 11975–11980.

Williams RH, Jensen LT, Verkhratsky A, Fugger L & Burdakov D (2007). Control of hypothalamic orexin neurons acid andCO2. Proc Natl Acad Sci U S A 104, 10685–10690.

Yamanaka A, Beuckmann CT, Willie JT, Hara J, Tsujino N,Mieda M et al . (2003a ). Hypothalamic orexin neuronsregulate arousal according to energy balance in mice. Neuron 38, 701–713.

Yamanaka A, Muraki Y, Tsujino N, Goto K & Sakurai T(2003b ). Regulation of orexin neurons by themonoaminergic and cholinergic systems. Biochem Biophys Res Commun 303, 120–129.

Ye JH, Wang F, Krnjevic K, Wang W, Xiong ZG & Zhang J(2004). Presynaptic glycine receptors on GABAergicterminals facilitate discharge of dopaminergic neurons inventral tegmental area. J Neurosci 24, 8961–8974.

Zhang LH, Gong N, Fei D, Xu L & Xu TL (2008a ). Glycineuptake regulates hippocampal network activity via glycinereceptor-mediated tonic inhibition.Neuropsychopharmacology 33, 701–711.

Zhang XB, Sun GC, Liu LY, Yu F & Xu TL (2008b ). α2 subunitspecificity of cyclothiazide inhibition on glycine receptors.

Mol Pharmacol 73, 1195–1202.

Author contributions

M.M.K. designed and performed most of the experiments and

data analysis, A.V. performed the perforated patch-experiments,

L.T.J. and L.F. generated and provided the transgenic mice,

D.B. obtained funding for the project, designed the study,and wrote the paper. All authors approved the final version.

The experiments were performed at the Department of

Pharmacology, University of Cambridge, UK.

Acknowledgements

This work was funded primarily by the European Research

Council (FP7 Grant to D.B.). M.M.K. was also supported by

Osk. Huttunen Foundation (PhD studentship).


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