639

J Physiol 589.3 (2011) pp 639651

Direct and indirect control of orexin/hypocretin neurons

by glycine receptors
Mahesh M. Karnani1 , Anne Venner1 , Lise T. Jensen2 , Lars Fugger3 and Denis Burdakov1
1

Department of Pharmacology, University of Cambridge, Cambridge, UK

Aarhus University Hospital, Aarhus, Denmark
3
Nuffield Department of Clinical Neurosciences, John Radcliffe Hospital, University of Oxford, Oxford, UK

The Journal of Physiology

Non-technical summary Normal wakefulness relies on brain cells called orexin/hypocretin

neurons. Activity of these cells stimulates awakening while their loss produces the sleep disorder
narcolepsy. By studying what makes orexin/hypocretin cells more or less active, we can thus gain
insights into how the brain switches between different states of consciousness. We describe a
new way to turn orexin/hypocretin cells off using a chemical called glycine. We show that glycine
shuts down the electrical activity of orexin/hypocretin neurons from the adult brain, but has the
opposite 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/hypocretin
neurons. These data shed new light on the basic chemical and physical mechanisms regulating
orexin/hypocretin neurons, which may also be useful in improving therapeutic strategies for
disorders such as insomnia.
Abstract Hypothalamic hypocretin/orexin (hcrt/orx) neurons promote arousal and reward
seeking, 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 understood. Here we show that glycine, a neurotransmitter best known for its actions in the brainstem
and spinal cord, elicits dose-dependent postsynaptic Cl currents in hcrt/orx cells in acute mouse
brain 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 functional
during both early postnatal and adult periods, and gramicidin-perforated patch-clamp recordings
revealed that they progressively switch from excitatory to inhibitory during the first two postnatal
weeks. The pharmacological profile of the glycine response suggested that developed hcrt/orx
neurons contain /-heteromeric GlyRs that lack 2-subunits, whereas 2-subunits are present
in early postnatal hcrt/orx neurons. All postsynaptic currents (PSCs) in developed hcrt/orx cells
were blocked by inhibitors of GABA and glutamate receptors, with no evidence of GlyR-mediated
PSCs. However, the frequency but not amplitude of miniature PSCs was reduced by strychnine
and increased by glycine in 50% of hcrt/orx neurons. Together, these results provide the first
evidence 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 located
on 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: dib22@cam.ac.uk and mmk37@cam.ac.uk
Abbreviations ACSF, artificial cerebrospinal fluid; eGFP, enhanced green fluorescent protein; GlyR, glycine receptor;
hcrt/orx, hypocretin/orexin; PSCs, postsynaptic currents.


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DOI: 10.1113/jphysiol.2010.198457

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M. M. Karnani and others

Introduction
Hypothalamic neurons that produce peptide transmitters
hypocretins/orexins (hereafter referred to as hcrt/orx
cells) are vital regulators of states of consciousness and
reward-seeking behaviour. Hcrt/orx cells are located in
the lateral hypothalamic area, but project widely to most
of the brain, where they excite target neurons through two
specific 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 sustained
consciousness that loss of orexin cells causes severe
narcolepsy/cataplexy (Thannickal et al. 2000; Hara et al.
2001). Hypocretins/orexins also stimulate feeding and
reward-seeking behaviour, and destruction of hcrt/orx
neurons impairs fasting-induced locomotor activity, and
leads to reduced energy expenditure and obesity (Hara
et 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 different
ways of manipulating hcrt/orx cell activity may thus help
design better treatment strategies for neurological and
psychiatric disorders.
The most common physiological way of constraining
the activity of a neural circuit is by activation of GABAA
receptors, which have been reported to be functional in
hcrt/orx cells (Li et al. 2002; Yamanaka et al. 2003b).
However, it is unknown how hcrt/orx cells are affected
by other fast transmitters that constrain neural activity,
such as glycine. Although glycine is best known as an
inhibitory neurotransmitter in the brainstem and spinal
cord (Werman et al. 1968; Gold & Martin, 1983), glycine
receptors (GlyRs) are also found in several higher brain
structures (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 produce
different effects (excitation or inhibition) depending on
the intracellular Cl concentration, which varies between
different cell types in adult brain (Tozuka et al. 2005; Choi
et al. 2008), as well as between different developmental
stages (Ben-Ari et al. 2007). How these factors affect
hcrt/orx neurons is unknown, because their responses to
glycine have not been examined, whereas their responses
to GABA have only been examined using whole-cell
recordings, where the intracellular Cl concentration is
artificially fixed.
Here, we study the electrical responses of identified
hcrt/orx neurons to glycine and other known modulators
of GlyRs. We find that hcrt/orx cells express functional

J Physiol 589.3

GlyRs from early postnatal stages through to adulthood.

The effect of activation of postsynaptic GlyR Cl channels
progressively changes from excitation to inhibition during
the development of the hcrt/orx network. In addition, in
developed hcrt/orx circuits, presynaptic GlyRs regulate
the release of both glutamate and GABA onto hcrt/orx
cells.
Methods
Preparation of living brain tissue

All animal procedures were performed in accordance

with the Animals (Scientific Procedures) Act 1986 UK,
following guidelines in Drummond (2009), and approved
by local animal welfare committees of the University of
Cambridge. Transgenic orexin-eGFP mice were used to
identify and study hcrt/orx neurons. These mice express
enhanced 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 lightdark cycle (lights
on at 08:00 h) and had free access to food and water.
Coronal slices 250 m thick containing the lateral hypothalamus were prepared from mice (ages as indicated in
the figure legends). Mice were killed by cervical dislocation
during the light phase and rapidly decapitated. Brains
were quickly removed and placed into ice-cold ACSF. A
block of brain tissue was glued to the stage of a Campden
Vibroslice for slicing while immersed in ice-cold ACSF.
After a 1 h recovery period at 35 C in ACSF, slices were
used 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 , 2
CaCl2 , 1.2 NaH2 PO4 , 21 NaHCO3 and 1 D-(+)-glucose.
For standard whole-cell recordings, three types of intracellular (pipette) solutions were used. High-Cl pipette
solution contained (in mM): 130 KCl, 0.1 EGTA, 10
Hepes, 5 K2 ATP, 1 NaCl, 2 MgCl2 , 40 sucrose, pH 7.3
with KOH. Low-Cl pipette solution contained (in
mM): 120 potassium gluconate, 10 KCl, 0.1 EGTA, 10
Hepes, 5 K2 ATP, 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, 0.1
EGTA, 10 Hepes, 5 K2 ATP, 1 NaCl, 2 MgCl2 , pH 7.3
with KOH. Liquid junction potentials for the low-Cl
and 43 mM Cl solutions were estimated to be 10.1
and 6.0 mV, respectively, and have been subtracted from
the measurements. For gramicidin-perforated whole-cell

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Glycine receptors in brain orexin circuits

recordings we filled pipettes with (in mM) 130 KCl, 0.1

EGTA, 10 Hepes, 5 K2 ATP, 1 NaCl, 2 MgCl2 , pH 7.3
with KOH and between 300 and 600 g ml1 gramicidin
(mix of A, B, C and D isoforms, Sigma). A stock solution
of 100 mg ml1 gramicidin was prepared in DMSO with
25 mg ml1 Pluronic F-127. The hypo-osmotic (30%
osmolarity) stimulation protocol shown in Fig. 5B was
based on Hussy et al. (1997), and consisted of switching
from a control solution (ASCF that contained 78 mM NaCl
and 94 mM sucrose) to a hypo-osmotic solution (same as
control solution but without sucrose).

Drugs

The following drugs were added to the extracellular

solution where indicated: 0.055 mM glycine (Sigma),
50 M (2R)-amino-5-phosphonovaleric acid (AP5),
10 M 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX),
10 M dizocilpine maleate (MK801), 50 or 100 M
picrotoxin (PiTX), 3 M gabazine, 0.0013 M strychnine,
1 M tetrodotoxin (TTX), 100 M cyclothiazide (CTZ)
and 5 mM L--alanine. All drugs were obtained from Sigma
or Tocris (UK). Chemicals were applied extracellularly
by bath superfusion. All drugs were dissolved in water
except PiTX and CTZ, which were dissolved in ethanol
and DMSO, respectively (0.1% final concentration).
Glutamatergic mPSCs were recorded in the presence
of 3 M gabazine and 1 M TTX, and verified as
glutamatergic by blockade with 10 M CNQX. GABAergic
mPSCs were recorded in the presence of 50 M AP5, 10 M
CNQX, 10 M MK801 and 1 M TTX, and verified as
GABAergic by blockade with 3 M gabazine.

Recording and analysis

Living orexin-eGFP neurons were visualized in brain

slices using an Olympus BX50WI upright microscope
equipped with oblique illumination optics, a mercury
lamp and filters for visualizing eGFP-containing cells.
Somatic recordings were carried out at 37 C using
an EPC 10 patch-clamp amplifier controlled by Pulse
and Patchmaster software (HEKA Elektronik, Germany).
Patch pipettes were made from borosilicate glass, and their
tip-resistances ranged from 3 to 8 M (35 M with
high-Cl and 58 M with low-Cl pipette solution).
Slices were placed in a submerged-type chamber (volume
2 ml, solution flow rate 2.5 ml min1 ) and anchored
with a nylon string grid stretched over platinum wire.
In standard whole-cell mode, only cells with access
resistances below 20 M were accepted for analysis.
In gramicidin-perforated mode, access resistances were
below 100 M. Signals were low-pass filtered at 3 kHz and
digitized at 7 kHz. Currentvoltage (IV ) relationships

641

shown in Figs 1D, 2D and 5B, were obtained by performing

voltage-clamp ramps from 10 to 140 mV at a rate of
0.1 mV ms1 .
To study evoked postsynaptic currents (Fig. 5A, right), a
concentric bipolar stimulation electrode (World Precision
Instruments) was placed within the lateral hypothalamus
50200 m away from the recorded cell. Stimulatory
pulse characteristics (100200 A, 0.2 ms, 0.2 Hz) were
controlled by a DS3 isolated stimulator (Digitimer, UK).
These responses were confirmed to be synaptic by blockade
with a cocktail of ionotropic glutamate, GABAA and
glycine receptor blockers at the end of each experiment.
During our analysis of mEPSCs, we looked at kinetics
of the individual synaptic events (using Minianalysis,
Synaptosoft, Fort Lee, NJ, USA), and confirmed that the
time constant of decay was <10 ms for all events analysed,
indicating that they were mediated by AMPA rather than
NMDA receptors (Dingledine et al. 1999), as expected
from the negative (60 mV) holding potential used in
these experiments.
Statistical analyses were performed using Minianalysis, Matlab (Mathworks, Natick, MA, USA) and
Origin (Microcal, Northampton, MA, USA) software.
Averaged data are presented as mean S.E.M. Statistical
significance was evaluated using Students t test unless
stated otherwise.
Data in Fig. 1C were fitted with the following equation:

I = I max

I max [strychnine]h
ICh50 + [strychine]h

where I max is the maximal current induced by 1 mM

glycine, IC50 is the concentration of strychnine that
produces half-maximal inhibition and h is the Hill
coefficient. The fit shown in Fig. 1C was obtained with
I max = 1360 pA, h = 0.91 and IC50 = 0.22 M.
Data in Figs 2A and 3A were fitted with the following
equation:

I =

I max [gly]h
ECh50 + [gly]h

where I max is the maximal current induced by glycine,

EC50 is the concentration that gives half-maximal response
and h is the Hill coefficient. The fit shown in Fig. 2A
was obtained with I max = 71.3 pA pF1 , h = 2.0 and
EC50 = 0.70 mM. The fit shown in Fig. 3A was obtained
with I max = 108.5 pA pF1 , h = 1.3 and EC50 = 0.44 mM.
The differences in I max , h and EC50 of these two
datasets did not reach statistical significance (P > 0.2,
F test).


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Results
GlyR modulators regulate postsynaptic Cl currents
in hcrt/orx neurons

To explore the effects of glycine on identified hcrt/orx cells,

we first analysed the effect of glycine on membrane current

J Physiol 589.3

using standard whole-cell voltage-clamp recordings from

identified hcrt/orx cells in acutely isolated mouse brain
slices. Glycine (0.5 mM) elicited large membrane currents
in all (>50) cells tested (Fig. 1A). The amplitudes of
glycine-induced currents were not significantly affected
by pharmacological synaptic isolation (Fig. 1A and B;

Figure 1. Biophysical properties of postsynaptic GlyRs in hcrt/orx neurons

Data in this figure are from P1327 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 M strychnine
completely blocked the response to 0.5 mM glycine (n = 5, see control trace shown in A for comparison). Right,
doseresponse of strychnine-induced inhibition of the current produced by 1 mM glycine (n > 3 cells per point,
IC50 = 0.22 M). D, net currentvoltage 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 M strychnine (n = 5). Values
are means (black) and S.E.M. (grey). E, dependence of the reversal potential of current activated by 0.5 mM glycine
on intracellular [Cl]; dashed line represents theoretical (Nernstian) Cl reversal potential, n = 5 for each point.

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Glycine receptors in brain orexin circuits

643

control, 790.4 91.5 pA at 60 mV, n = 7 cells; synaptic

blockers, 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 currentvoltage relationship of the
glycine-activated current exhibited outward rectification
(Fig. 1D, n = 11), in agreement with known biophysical
properties of GlyR Cl channels (Rajendra et al. 1997). As
the intracellular chloride concentration was progressively
reduced, the reversal potential of the glycine-activated
current became progressively more negative, in good
agreement with the Nernst prediction for a Cl -selective
ion channel (Fig. 1D and E). Together, these data
strongly imply that hcrt/orx cells express functional
strychnine-sensitive GlyRs.
To functionally characterize the type of GlyR expressed
by hcrt/orx neurons, we examined the doseresponse
relationship of glycine activation of membrane currents.
Bath application of 50 M to 5 mM glycine induced
dose-dependent responses with EC50 of 0.7 mM (Fig. 2A).
Application of 100 M picrotoxin, which is expected
to block -homomeric GlyRs but not /-heteromeric
GlyRs (Pribilla et al. 1992), did not reduce the amplitude
of 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 GlyRs
are involved. Application of 100 M cyclothiazide,
a selective blocker of 2-containing GlyRs (Zhang
et al. 2008b), also did not affect the amplitude of
glycine-induced currents (Fig. 2B; at 60 mV, cyclothiazide = 746.8 101.5 pA; control 803.0 179.4 pA,
n = 4, P > 0.5), arguing against the presence of 2 GlyRs
in developed hcrt/orx cells. Like glycine, L--alanine,
another GlyR agonist (Rajendra et al. 1997), elicited
strychnine-sensitive, Cl -selective, outwardly rectifying
membrane currents (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).

Figure 2. Pharmacological properties of postsynaptic GlyRs in

hcrt/orx neurons

Data in this figure are from P1327 mice. A, doseresponse 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 M, n = 3). The reversal potential was 63.3 3.7 mV
in 15 mM intracellular Cl (predicted Nernst ECl = 58.8 mV). Values
are means (black) and S.E.M. (grey).


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Responses of hcrt/orx neurons to glycine change

during development

The above experiments used postnatal day (P) 1327

mice. To examine developmental properties of hcrt/orx
cell GlyRs, we also studied neonatal (P35) mice. The
neonatal hcrt/orx cells also displayed dose-dependent
glycine currents (Fig. 3A), and tended to be slightly more
sensitive to glycine (EC50 = 0.44 mM) than mature cells
(EC50 = 0.7 mM; see Fig. 2A), although the difference in
sensitivity did not reach statistical significance (P > 0.3,
F test). Application of 100 M picrotoxin or 100 M
cyclothiazide significantly reduced the amplitude of
glycine-induced currents in P35 hcrt/orx cells (Fig. 3B
and C, statistics for raw data and normalized responses are
given in the figure legend). This suggests that, in contrast to
P1327 hcrt/orx cells where picrotoxin and cyclothiazide
were ineffective (Fig. 2B), neonatal hcrt/orx cells contain
some -homomeric GlyRs (as implied by picrotoxin
sensitivity) and 2-containing GlyRs (as implied by cyclothiazide sensitivity).
To determine the physiological effect (depolarization
vs. hyperpolarization) of glycine-induced currents in
hcrt/orx cells, we monitored the membrane potential
using gramicidin-perforated patch recordings, which
preserve the endogenous Cl concentration in hcrt/orx
cell cytosol, and so allow GlyR Cl channels to exert their
true physiological effects on the membrane potential. In
gramicidin-perforated patch recordings, glycine elicited
robust 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 analysed
at each time point). We confirmed that these differences
(hyperpolarizing vs. depolarizing) were not due to
differences in resting membrane potentials (RMP) in
glycine-hyperpolarized and glycine-depolarized cells, and
thus probably resulted from developmental changes
in the transmembrane Cl gradient (RMP of hyperpolarized cells was 47.0 1.0 mV, RMP of depolarized
cells was 45.8 1.6 mV, n = 22 and 12, respectively,
P > 0.3). We also carried out control experiments in the

Figure 3. Properties of glycine-induced currents in neonatal

(P35) hcrt/orx cells

A, doseresponse 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 P35 and P1420 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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645

presence of the NMDA receptor blocker AP5 (50 M),

which confirmed that glycine action on the NMDA
receptor (Dingledine et al. 1999) did not contribute to
our results (Fig. 4B, 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). Our
analysis of cells from P3 to P19 indicated that the
switch from depolarizing to hyperpolarizing responses
to glycine is completed between P15 and P19 (Fig. 4C).
These results probably reflect a gradual development
of the transmembrane Cl gradient in hcrt/orx cells
during the first weeks of life (see Discussion). Finally, we
also confirmed that strychnine-sensitive glycine currents
were present in old (P140) mice (current induced by
0.5 mM glycine without strychine = 43.7 5.6 pA pF1 ;
with 3 M strychnine = 0.2 0.1 pA pF1 ; P < 0.001,
n = 4, holding potential = 60 mV, data not shown),
indicating that hcrt/orx cells can be controlled by GlyRs
throughout the animals 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, the
remaining sPSCs had a frequency of 1.01 0.19 Hz
(Fig. 5A, n = 5 cells). However, these sPSCs were
exclusively GABAergic, with no contributions from postsynaptic GlyRs, because they were completely abolished
by 3 M gabazine (Fig. 5A), which blocks GABAergic
currents without affecting glycinergic currents (Chery
& de Koninck, 1999; Mori et al. 2002; Beato, 2008).
This suggests that all spontaneous synaptic currents
observed in hcrt/orx cells are mediated by glutamate
and GABA receptors. We also analysed the amplitude
of evoked PSCs (ePSCs, see Methods), and found it
unaffected by strychnine (Fig. 5A; ePSC amplitude in
strychnine was 100.3 8.6% of control, n = 4, P > 0.5).
This confirms that postsynaptic GlyRs on hcrt/orx cells
are not activated by synaptic release (see Discussion).
Apart from synaptically released glycine, GlyRs could also
be activated by taurine released from astrocytes under
hypo-osmotic conditions (Hussy et al. 2000). However, we
found that membrane potential and membrane current
responses of hcrt/orx neurons to a hypo-osmotic stimulus
(30% reduction in osmolarity, see Methods) did not have
a strychnine-sensitive component (Fig. 5B, membrane
potential data: control response = 19.7 1.3 mV
depolarization; strychnine response = 21.4 2.9 mV
depolarization; P > 0.5; membrane current data: at
60 mV, 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 effects
described in the previous 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 by
the 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 P1327 mice. Left
(top), an example of voltage-clamp recording in the
presence 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 M 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 M strychnine
(amplitude in strychnine was 100.3 8.6% of
control, n = 4 cells, P > 0.5). B, data in this panel
are from P1327 mice. Left, membrane potential
responses to hypo-osmolarity (see Methods) in the
absence (top) and presence (bottom) of 3 M
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 M
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 P2227 mice. Examples
of GABA mPSCs in the presence and absence of
1 M strychnine (recorded with 50 M AP5, 10 M
CNQX, 10 M MK801 and 1 M TTX in bath,
60 mV holding potential, high-Cl pipette
solution). D, data in this panel are from P2227
mice. Left, inter-event intervals (IEI) of GABA mPSCs
from 4 out of 9 cells that responded to 1 M
strychnine (control, continuous line; strychnine,
dashed line). KolmogorovSmirnov 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 KolmogorovSmirnov test; inset shows
means (strychnine decreased amplitude by
1.1 4.0% relative to control, P > 0.8).


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J Physiol 589.3

Glycine receptors in brain orexin circuits

glutamate and GABA terminals contacting hcrt/orx cells,

we blocked action potential-mediated synaptic release
with tetrodotoxin, and examined the frequency of the
resulting miniature post-synaptic currents (mPSCs).
We found that strychnine significantly decreased the
frequency, but did not affect the amplitude, of GABAergic
mPSCs in 4 out of 9 cells tested (Fig. 5C and D, statistics
are given in legend to Fig. 5D), suggesting that presynaptic GlyRs tonically enhance the release of GABA
onto hcrt/orx cells. Surprisingly, we also observed that
strychnine had a significant inhibitory effect on the
frequency (but not amplitude) of glutamatergic mPSCs in
4 out of 9 cells tested (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-clamp
the postsynaptic cells near the reversal potential of
GlyR-mediated responses, because we found that at
other holding potentials, the large postsynaptic channel
noise induced by glycine (Fig. 1A) obscured the mPSCs.
It was thus only possible to examine the effects on
glutamate mPSCs, because GABA mPSCs reverse at
the same potential as glycine currents and thus are
invisible with this voltage-clamp protocol (60 mV
holding potential, low-Cl pipette solution). We found
that glycine significantly increased the frequency (but
not amplitude) of glutamate mPSCs in 4 out of 7 cells
tested (Fig. 7, statistics are given in legend to Fig. 7B),
providing further evidence for the existence of a
presynaptic population of GlyRs.

647

We found that GlyR agonists triggered large postsynaptic Cl currents in hcrt/orx cells. This response
was directly mediated by GlyRs on the recorded
neuron, as shown by lack of blockade by TTX and
blockers of ionotropic GABA and glutamate receptors,

Discussion
Although the importance of GlyRs in brainstem and
spinal cord is well established (Rajendra et al. 1997),
the function of GlyRs in higher brain areas is less
understood. 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 hypothalamic
neurons remained largely unknown. This is the first report
linking modulation of glycine receptors to the activity
of identified hcrt/orx cells, key hypothalamic players in
the regulation of wakefulness, energy expenditure and
reward seeking. Our results provide evidence that the
activity of hcrt/orx cells is regulated by functional GlyRs
located on both postsynaptic sites on the hcrt/orx cell
membrane and on glutamatergic and GABAergic synaptic
terminals contacting hcrt/orx cells. Since the action of
glycine on GlyRs on mature hcrt/orx cells was hyperpolarizing, whereas the action on presynaptic GlyRs
increased synaptic release onto hcrt/orx neurons, these
data reveal two distinct mechanisms for modulating the
firing of hcrt/orx neurons.

Figure 6. Presynaptic GlyRs tonically enhance glutamate

release onto hcrt/orx neurons
Data in this figure are from P2227 mice. A, examples of glutamate
mPSCs in the presence and absence of 1 M strychnine (recorded
with 3 M gabazine and 1 M 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 M
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 KolmogorovSmirnov test; inset shows means (strychnine
increased amplitude by 3.2 2.2% relative to control, P > 0.1).


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M. M. Karnani and others

Figure 7. Glycine enhances glutamate release onto hcrt/orx

neurons
Data in this panel are from P1927 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-event
intervals (IEI) of glutamate mPSCs from 4/7 cells that responded to
0.5 mM glycine (control, continuous line; strychnine, dashed line).
KolmogorovSmirnov 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
KolmogorovSmirnov test; inset shows means (glycine decreased
amplitude by 1.4 1.7% relative to control, P > 0.2).

J Physiol 589.3

and by blockade by strychnine. Our comparison of

agonist and antagonist potencies suggested that hcrt/orx
cell GlyRs undergo a change in subunit composition
during development. Neonatal hcrt/orx cells appeared to
contain a significant proportion of -homomeric and
2-containing GlyRs, as implied by cyclothiazide and
picrotoxin sensitivity of glycine responses. In contrast, the
glycine responses of mature hcrt/orx cells were insensitive
to cyclothiazide and picrotoxin, suggesting that they
contain heteromeric GlyR channels that lack 2 subunits.
This is consistent with other studies (e.g. Malosio et al.
1991) suggesting that the expression of 2 subunits
decreases after birth. Although our glycine EC50 values
could 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 2-containing GlyRs
(EC50 300 M, Grenningloh et al. 1990; Schmieden et al.
1992), toward 3-containing GlyRs (EC50 0.75 mM,
Kuhse et al. 1990), rather than 1-containing GlyRs of
200 M, (Lewis et al. 1998).
Although in some neurocircuits GlyRs are
developmentally down- or up-regulated during the
first few postnatal weeks (Malosio et al. 1991; Turecek
& Trussell, 2002; Kubota et al. 2010) and seem to have
developmental roles (Flint et al. 1998), our data indicate
that functional postsynaptic GlyRs are present on orexin
neurons well into adulthood. While this does not preclude
a developmental role for the GlyRs, it does imply that they
serve some function in the adult. Gramicidin-perforated
patch-clamp recordings revealed that when [Cl ]i was
unperturbed, most orexin neurons hyperpolarized in
response to glycine application after P15, whereas between
P3 and P10 around half of the cells were depolarized by
glycine. The time-line of Cl gradient maturation, which
is thought to result from developmental up-regulation
of plasmalemmal K+ /Cl cotransporter KCC2 (Rivera
et al. 1999), was similar to that previously reported in
the hypothalamus (Gao & van den Pol, 2001; Wang
et al. 2001) and in other brain regions (Ben-Ari et al.
2007).
We did not observe any endogenous glycine-mediated
synaptic currents in hcrt/orx cell membrane, suggesting
that synapses contacting hcrt/orx cells do not release
glycine, and modulators of the postsynaptic GlyRs in
hcrt/orx cells are likely to come from extrasynaptic
sources. For example, the levels of glycine and alanine
in the extracellular space change during fasting and
feeding. During prolonged fasting in man, the plasma
concentration of alanine falls significantly within 3 days
whereas glycine rises within 10 days (Adibi, 1968; Felig
et al. 1969). Interestingly, Adibi (1968) also showed that
during isocaloric protein-free dieting, plasma alanine is
robustly elevated. As changes in intragastric levels of
amino acids subsequently lead to robust changes in amino
acid levels in the lateral hypothalamus (Choi et al. 1999),

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Glycine receptors in brain orexin circuits

it is possible that under certain circumstances glycine

(and/or alanine) may tonically regulate hcrt/orx neurons
via extrasynaptically located GlyRs, as proposed for some
other central neurons (Flint et al. 1998; Mori et al.
2002; Meier et al. 2005; Zhang et al. 2008a). Another
endogenous ligand of GlyRs is taurine, which in some
hypothalamic regions may be released from astrocytes
under hypo-osmotic conditions (Hussy et al. 2001).
However, we found that responses of hcrt/orx neurons
to hypo-osmolarity did not have a strychnine-sensitive
component, arguing against this mechanism in hcrt/orx
cells.
In terms of effects of GlyR modulation on endogenous
synaptic 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 and
glutamate mPSCs, while GlyR activation increased the
frequency of glutamate mPSCs (we could not measure
effects of glycine on GABA mPSCs for technical reasons
described above). Because mPSC frequency is a standard
measure of presynaptic release probability, the simplest
explanation for these results is that there are excitatory
GlyRs located on glutamate and GABA synaptic terminals
contacting hcrt/orx cells, which are tonically active in
our brain slice preparation. We note that, although
the classical postsynaptic action of glycine is hyperpolarizing and inhibitory, there is evidence from other
preparations that GlyRs located on presynaptic nerve
terminals 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; Lee
et al. 2009). Our data suggest that similar excitatory
GlyRs operate in both GABA and glutamate terminals
contacting hcrt/orx neurons. Presumably the synapses
containing presynaptic GlyRs comprise only a small
fraction of total synapse number on hcrt/orx cells, since
we could not resolve a significant effect of strychnine
on responses involving synchronized activation of many
synapses (Fig. 5A, right trace). It remains to be determined
whether presynaptic GlyRs on the glutamate and GABA
terminals are activated separately or together, and we
can 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 1980s
points to an existence of an inhibitory glycinergic
corticohypothalamic pathway (Kita & Oomura, 1981,
1982).
In summary, the functional GlyRs in hcrt/orx networks
represent a previously undescribed way to control the
activity of the hcrt/orx system, and could potentially be
engaged by GlyR-modulating drugs (Nguyen et al. 2009),
and as yet undefined physiological modulators, to control
brain function.

649

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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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