Activation of NPY type 5 receptors induces a long-lasting increase in
spontaneous GABA release from cerebellar inhibitory interneurons
C. J. Dubois,1,2P. Ramamoorthy,2M. D. Whim,1,2and S. J. Liu1,2
1Department of Cell Biology and Anatomy, Louisiana State University Health Sciences Center, New Orleans, Louisiana; and
2Department of Biology, Pennsylvania State University, State College, Pennsylvania
Submitted 17 August 2011; accepted in final form 20 December 2011
Dubois CJ, Ramamoorthy P, Whim MD, Liu SJ. Activation of
NPY type 5 receptors induces a long-lasting increase in spontaneous
GABA release from cerebellar inhibitory interneurons. J Neuro-
physiol 107: 1655–1665, 2012. First published December 21, 2011;
doi:10.1152/jn.00755.2011.—Neuropeptide Y (NPY), a widely dis-
tributed neuropeptide in the central nervous system, can transiently
suppress inhibitory synaptic transmission and alter membrane excit-
ability via Y2 and Y1 receptors (Y2rs and Y1rs), respectively. Al-
though many GABAergic neurons express Y5rs, the functional role of
these receptors in inhibitory neurons is not known. Here, we investi-
gated whether activation of Y5rs can modulate inhibitory transmission
in cerebellar slices. Unexpectedly, application of NPY triggered a
long-lasting increase in the frequency of miniature inhibitory post-
synaptic currents in stellate cells. NPY also induced a sustained
increase in spontaneous GABA release in cultured cerebellar neurons.
When cerebellar cultures were examined for Y5r immunoreactivity,
the staining colocalized with that of VGAT, a presynaptic marker for
GABAergic cells, suggesting that Y5rs are located in the presynaptic
terminals of inhibitory neurons. RT-PCR experiments confirmed the
presence of Y5r mRNA in the cerebellum. The NPY-induced poten-
tiation of GABA release was blocked by Y5r antagonists and mim-
icked by application of a selective peptide agonist for Y5r. Thus Y5r
activation is necessary and sufficient to trigger an increase in GABA
release. Finally, the potentiation of inhibitory transmission could not
be reversed by a Y5r antagonist once it was initiated, consistent with
the development of a long-term potentiation. These results indicate
that activation of presynaptic Y5rs induces a sustained increase in
spontaneous GABA release from inhibitory neurons in contrast to the
transient suppression of inhibitory transmission that is characteristic
of Y1r and Y2r activation. Our findings thus reveal a novel role of
presynaptic Y5rs in inhibitory interneurons in regulating GABA
release and suggest that these receptors could play a role in shaping
neuronal network activity in the cerebellum.
neuropeptide Y; ?-aminobutyric acid; cerebellum; inhibitory GABAergic
NEUROPEPTIDE Y (NPY) is the most abundant neuropeptide in
the brain. Although NPY is thought to regulate arousal (Fu et
al. 2004) and feeding behavior (Chee and Colmers 2008),
increasing evidence indicates that it can also play an important
role in modulating emotional states, such as anxiety, depres-
sion, and fear (Karlsson et al. 2005; Morales-Medina et al.
2010). These diverse physiological actions presumably arise
from the expression of NPY in multiple brain regions
(Akiyama et al. 2008; Morin and Gehlert 2006) where both
neurons and glial cells can synthesize and release NPY (Ra-
mamoorthy and Whim 2008; Shinoda et al. 1989; Ubink et al.
2003). The repertoire of NPY actions is further expanded by a
family of NPY receptors (Y1–y6). For example, Y1 and Y2
receptors (Y1rs and Y2rs) are known to reduce membrane
excitability and transiently suppress neurotransmitter release,
respectively (Sun et al. 2001a). However, the functional role of
Y5rs remains largely elusive.
Y5rs have been postulated to have anxiolytic actions (So-
rensen et al. 2004) and to suppress epileptiform seizures (Guo
et al. 2002; Woldbye et al. 2005). Therefore, it would seem
possible that activation of Y5rs could enhance inhibitory trans-
mission, thus suppressing network activity, particularly since
Y5rs are preferentially expressed in a subset of GABAergic
interneurons in several brain regions (Campbell et al. 2001;
Grove et al. 2000).
To test this idea, we have examined the ability of NPY to
regulate transmission between cerebellar interneurons. Inhibi-
tory synaptic transmission controls cerebellar output by tuning
the excitability of Purkinje cells and is required to optimize the
cerebellar learning process. Genetic deletion of GABA recep-
tors on Purkinje cells leads to a deficit in the consolidation of
vestibulocerebellar motor learning (Wulff et al. 2009). There-
fore, GABA release from cerebellar interneurons is expected to
be tightly regulated by neuronal activity. Indeed, fear condi-
tioning induces a sustained increase in GABA release from
inhibitory interneurons (Scelfo et al. 2008), and we have re-
cently shown that parallel fiber activation triggers a lasting
enhancement in GABA release from stellate cells (Lachamp et
al. 2009). Given that NPY is expressed in two major inputs to
the cerebellum, climbing and mossy fibers (Laemle et al. 1991;
Ueyama et al. 1994), NPY is a likely modulator of cerebellar
inhibitory synaptic transmission.
Here, we show that cerebellar inhibitory interneurons ex-
press presynaptic Y5rs. NPY application was found to induce
a long-lasting increase in spontaneous GABA release in con-
trast to the NPY-induced transient suppression of GABA re-
lease observed in several brain regions (Chen and van den Pol
1996; Sun et al. 2001a). The induction by NPY of the long-
term potentiation of inhibitory synapses (I-LTP) was com-
pletely abolished by Y5r antagonists and mimicked by appli-
cation of a Y5r agonist. Furthermore, Y5r immunoreactivity
(-ir) colocalized with that of vesicular GABA transporter
(VGAT), suggesting that activation of presynaptic Y5rs trig-
gers the sustained enhancement of spontaneous GABA release.
Our findings reveal a novel function for Y5rs at the presynaptic
terminals of inhibitory neurons, namely the induction of a
long-lasting increase in GABA release. These receptors may
therefore contribute to the suppression of neuronal network
Address for reprint requests and other correspondence: S. J. Liu, Dept. of
Cell Biology and Anatomy, LSU Health Sciences Center, 1901 Perdido St.,
New Orleans, LA 70112 (e-mail: email@example.com).
J Neurophysiol 107: 1655–1665, 2012.
First published December 21, 2011; doi:10.1152/jn.00755.2011.
16550022-3077/12 Copyright © 2012 the American Physiological Societywww.jn.org
Animals. We used postnatal day 5 (P5)-to-P7 (for cell culture) and
P21-to-P24 (for slice electrophysiology) C57BL/6J mice (The Jack-
son Laboratory) bred and housed in our facility on a 12:12-h light-
dark cycle. All experimental procedures were approved by the Animal
Care and Use Committee of Louisiana State University Health Sci-
ences Center and of the Pennsylvania State University.
Cerebellar slice preparation and electrophysiology. Cerebellar slices
were prepared from 3-wk-old mice as previously described (Liu and
Cull-Candy 2000). Briefly, mice were decapitated, and the cerebellum
was quickly isolated before slicing. Sagittal slices (300 ?m) were cut
from the vermis of the cerebellum using a microslicer (Leica VT1200)
in ice-cold artificial cerebrospinal fluid (ACSF) that contained 7 mM
MgCl2and 1 mM CaCl2. Slices were then maintained in ACSF (in
mM: 125 NaCl, 2.5 KCl, 26 NaHCO3, 1.25 NaH2PO4, 1 MgCl2, 2
CaCl2, and 25 glucose, pH 7.4) saturated with 95% O2-5% CO2at
room temperature for 30 min before recording.
Whole cell patch-clamp recordings were made at near physiolog-
ical temperature (33–36°C) in O2-CO2-bubbled ACSF. Stellate cells
in the molecular layer of lobules V and VI were identified by their
location in the outer two-thirds of the molecular layer and by the
presence of spontaneous action potentials in a cell-attached mode.
Synaptic currents were recorded when stellate cells were voltage-
clamped at ?60 mV (MultiClamp 700A; Axon Instruments) using
borosilicate electrodes (6–8 M?) filled with a cesium-based internal
solution (in mM: 130 CsCl, 2 NaCl, 1 CaCl2, 4 MgATP, 10 Cs-
EGTA, 1 QX-314, 5 tetraethylammonium, and 10 HEPES, pH 7.25).
Miniature inhibitory postsynaptic currents (mIPSCs) were recorded in
the presence of 5 ?M 2,3-dihydro-6-nitro-7-sulfamoyl-benzo(f)qui-
noxaline (NBQX) and 0.5 ?M TTX in the extracellular solution.
IPSCs were filtered at 6 kHz and digitized at 20 kHz. Series resistance
was monitored throughout all the experiments. If series resistance
changed ?20%, recordings were terminated. Data analysis was per-
formed with Clampfit 9.0 software (Axon Instruments) using the
built-in event detection template.
Cerebellar cell culture and electrophysiology. Cerebella of 1-wk-
old mice were dissociated as described previously (Fiszman et al.
2005). Briefly, cerebella were treated with trypsin (1.4 mg/ml) and
plated on poly-D-lysine-coated (0.1 ?g/ml) glass coverslips in Eagle’s
basal medium (without glutamine, supplemented with 10% fetal
bovine serum). On day 1 in vitro (DIV1), half of the culture medium
was replaced by serum-free neurobasal medium (without glutamine,
supplemented with B27). On DIV5, cytosine arabinoside (5 ?M) was
added. Cultures were kept at 37°C for 11–21 days.
Whole cell patch-clamp recordings were made at 22°C from
cultured granule cells in an extracellular solution (in mM: 145 NaCl,
3 KCl, 1 MgCl2, 2 CaCl2, 25 glucose, and 10 HEPES, pH 7.3).
Granule cells were identified by their morphological characteristics
and lack of spontaneous action potentials in cell-attached mode.
mIPSCs were recorded at ?60 mV in the presence of NBQX and TTX
using a cesium-based internal solution (see above). Series resistance
was also monitored, and the same rejection criteria were applied.
Immunocytochemistry. Cells were fixed and permeabilized in 100%
acetone at ?20°C. Immunostaining of cerebellar cultured neurons
was performed largely as described previously (Ramamoorthy et al.
2011). Primary antibodies used were rabbit anti-Y1r (1:100; Immu-
noStar), rabbit anti-Y5r (1:100; Sigma-Aldrich Prestige Antibodies),
and guinea pig anti-VGAT (1:1,000; Synaptic Systems) and anti-
vesicular glutamate transporter (anti-vGlut; 1:1,000; Synaptic Sys-
tems). Secondary antibodies used were donkey anti-guinea pig FITC
(1:50; Jackson ImmunoResearch) and donkey anti-rabbit DyLight 549
(1:100; Jackson ImmunoResearch). Double-staining experiments
were conducted by applying the antibodies sequentially with the Y5r
antibody applied first. Control experiments showed no detectable
cross-reactivity between antibodies.
Imaging. Images were acquired with a Leica TCS SP2 SE Confocal
Microscope (?10 dry and ?60 water immersion objectives; Leica
Microsystems) using Leica Confocal Software (version 6.2).
Cell transfection. INS-1 832/13 cells, a clonal ?-cell line, were
maintained in RPMI containing 10% FCS, 50 ?M 2-mercaptoetha-
nol, 1 mM sodium pyruvate, and 1 mM HEPES as previously
described (Whim 2011). Cells were plated and 48 h later cotrans-
fected with 0.2 ?g of green fluorescent protein (GFP) and 1.3 ?g
of human Y1r, Y2r, or Y5r cDNA in pcDNA3.1 (Missouri S&T
cDNA Resource Center) using Lipofectamine 2000 (Invitrogen).
Cells were used for immunostaining 2 days later. AtT20 cells were
cultured as described in Mitchell et al. (2008) and cotransfected
with GFP and human Y2r cDNA.
RT-PCR. Total RNA was isolated from cerebellum and whole brain
of adult (?P21) mice using TRIzol (Invitrogen) as described in
Ramamoorthy and Whim (2008). RNA was purified using RNeasy
(Qiagen), and 2 ?g was used for reverse transcription in a volume of
50 ?l. Subsequent PCR reactions used 4 ?l of the template cDNAs in
a total volume of 50 ?l. The hot-start PCR protocol was 35 cycles
(94°C, 45 s; 55°C, 45 s; 72°C, 60 s), and products (20 ?l) were run
on 2% agarose gels. Primers were:
Y1; 5=-CGGCGTTCAAGGACAAGTAT-3= and 5=-TGATTCGC-
TTGGTCTCACTG-3=; 216-bp product, 326 genomic.
Y2; 5=-TGCCAATCTGGTTAGGGAAG-3= and 5=-GGTGCCAA-
CTCCTTGTTCTG-3=; 233-bp product.
Y4; 5=-CTGGCCCAAAAGTCTTCATC-3= and 5=-CTCCCAGC-
ACCTGCTTCTAC-3=; 129-bp product.
Y5; 5=-CAGATTAATCCAGCTGTTCTGC-3= and 5=-GAAAAC-
AGCCTTTATTTGACAATG-3=; 111-bp product.
y6; 5=-TCACTAAATAAGACCATCGGGTAG-3= and 5=-GGGA-
GGTTTACCCTAGGAAATG-3=; 126-bp product.
NPY; 5=-GCTAGGTAACAAGCGAATGGGG-3= and 5=-CACA-
TGGAAGGGTCTTCAAGC-3=; 288-bp product, 5724 genomic. Y2,
Y4, Y5, and y6 primers were previously described (Klenke et al.
Data analysis. Results are presented as means ? SE. Signifi-
cance was assessed by a two-tailed Student’s t-test or one-/two-
way ANOVAs followed by a Tukey post hoc test. Values of P ?
0.05 were considered significant.
NPY induces an increase in GABA release from inhibitory
neurons in cerebellar slices and cultures. Cerebellar stellate
cells in the molecular layer innervate each other. We thus re-
corded mIPSCs from these neurons to monitor spontaneous
GABA release from other stellate cells in cerebellar slices. To
examine the impact of NPY application on spontaneous GABA
release from cerebellar inhibitory neurons, we determined the
frequency of mIPSCs before and following the application of
NPY in acute cerebellar slices at 33–36°C (Fig. 1). After
obtaining a stable recording of mIPSCs for 20 min, 1 ?M NPY
was applied for 15 min. mIPSC frequency started to increase
during NPY application (control, 0.4 ? 0.1 Hz; NPY, 0.5 ?
0.1 Hz; n ? 7; paired t-test, P ? 0.05) and reached a plateau
of 104 ? 39% potentiation 15–30 min after NPY application
(0.7 ? 0.0 Hz; paired t-test, P ? 0.01 vs. control; Fig. 1C). The
mIPSC frequency remained elevated for at least 30 min in all
cells recorded. Thus NPY induced a sustained increase in
spontaneous GABA release from inhibitory neurons [ANOVA
test(time), P ? 0.05; Fig. 1C]. In contrast, the amplitude re-
mained unaltered during and following NPY applica-
tion [control, 154 ? 27 pA; 15–30 min after NPY application,
142 ? 30 pA; n ? 7; ANOVA test(time), P ? 0.05; Fig. 1C]. A
slow increase in decay time of mIPSCs [control, 5.9 ? 0.7 ms;
1656NPY ENHANCES GABA RELEASE THROUGH Y5 RECEPTORS
J Neurophysiol • doi:10.1152/jn.00755.2011 • www.jn.org
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1665NPY ENHANCES GABA RELEASE THROUGH Y5 RECEPTORS
J Neurophysiol • doi:10.1152/jn.00755.2011 • www.jn.org