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. 2011 Dec 21;107(6):1655–1665. doi: 10.1152/jn.00755.2011

Activation of NPY type 5 receptors induces a long-lasting increase in spontaneous GABA release from cerebellar inhibitory interneurons

C J Dubois 1,2, P Ramamoorthy 2, M D Whim 1,2, S J Liu 1,2,✉
PMCID: PMC3311673  PMID: 22190627

Abstract

Neuropeptide Y (NPY), a widely distributed neuropeptide in the central nervous system, can transiently suppress inhibitory synaptic transmission and alter membrane excitability via Y2 and Y1 receptors (Y2rs and Y1rs), respectively. Although many GABAergic neurons express Y5rs, the functional role of these receptors in inhibitory neurons is not known. Here, we investigated 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 postsynaptic 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 potentiation of GABA release was blocked by Y5r antagonists and mimicked 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.

Keywords: neuropeptide Y, γ-aminobutyric acid, cerebellum, inhibitory GABAergic interneurons

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, depression, 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 (Ramamoorthy 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 (Sorensen 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 transmission, 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. Inhibitory 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 receptors on Purkinje cells leads to a deficit in the consolidation of vestibulocerebellar motor learning (Wulff et al. 2009). Therefore, GABA release from cerebellar interneurons is expected to be tightly regulated by neuronal activity. Indeed, fear conditioning induces a sustained increase in GABA release from inhibitory interneurons (Scelfo et al. 2008), and we have recently 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 express presynaptic Y5rs. NPY application was found to induce a long-lasting increase in spontaneous GABA release in contrast to the NPY-induced transient suppression of GABA release 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 completely abolished by Y5r antagonists and mimicked by application of a Y5r agonist. Furthermore, Y5r immunoreactivity (-ir) colocalized with that of vesicular GABA transporter (VGAT), suggesting that activation of presynaptic Y5rs triggers 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 activity.

METHODS

Animals.

We used postnatal day 5 (P5)-to-P7 (for cell culture) and P21-to-P24 (for slice electrophysiology) C57BL/6J mice (The Jackson 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 Sciences 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 μm) were cut from the vermis of the cerebellum using a microslicer (Leica VT1200) in ice-cold artificial cerebrospinal fluid (ACSF) that contained 7 mM MgCl2 and 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% CO2 at room temperature for 30 min before recording.

Whole cell patch-clamp recordings were made at near physiological 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 MΩ) 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 μM 2,3-dihydro-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX) and 0.5 μM 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 performed 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 μg/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 μM) 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; ImmunoStar), 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 Systems). 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 β-cell line, were maintained in RPMI containing 10% FCS, 50 μM 2-mercaptoethanol, 1 mM sodium pyruvate, and 1 mM HEPES as previously described (Whim 2011). Cells were plated and 48 h later cotransfected with 0.2 μg of green fluorescent protein (GFP) and 1.3 μg 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 μg was used for reverse transcription in a volume of 50 μl. Subsequent PCR reactions used 4 μl of the template cDNAs in a total volume of 50 μl. The hot-start PCR protocol was 35 cycles (94°C, 45 s; 55°C, 45 s; 72°C, 60 s), and products (20 μl) were run on 2% agarose gels. Primers were:

Y1; 5′-CGGCGTTCAAGGACAAGTAT-3′ and 5′-TGATTCGCTTGGTCTCACTG-3′; 216-bp product, 326 genomic.

Y2; 5′-TGCCAATCTGGTTAGGGAAG-3′ and 5′-GGTGCCAACTCCTTGTTCTG-3′; 233-bp product.

Y4; 5′-CTGGCCCAAAAGTCTTCATC-3′ and 5′-CTCCCAGCACCTGCTTCTAC-3′; 129-bp product.

Y5; 5′-CAGATTAATCCAGCTGTTCTGC-3′ and 5′-GAAAACAGCCTTTATTTGACAATG-3′; 111-bp product.

y6; 5′-TCACTAAATAAGACCATCGGGTAG-3′ and 5′-GGGAGGTTTACCCTAGGAAATG-3′; 126-bp product.

NPY; 5′-GCTAGGTAACAAGCGAATGGGG-3′ and 5′-CACATGGAAGGGTCTTCAAGC-3′; 288-bp product, 5724 genomic. Y2, Y4, Y5, and y6 primers were previously described (Klenke et al. 2010).

Data analysis.

Results are presented as means ± SE. Significance 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.

RESULTS

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 recorded 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 μM 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 remained unaltered during and following NPY application [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; 15–30 min after NPY application, 7.0 ± 0.5 ms; n = 7; ANOVA test(time), P < 0.05] was unlikely due to an effect of NPY application since it was also observed in control [0–15 min, 7.3 ± 0.4 ms; 45 min later, 9.7 ± 0.9 ms; n = 9; ANOVA test(time), P < 0.05, interaction in 2-way ANOVA(time × drug application), P > 0.05]. These results suggest that NPY may act at the presynaptic terminals of inhibitory neurons to trigger an increase in spontaneous GABA release.

Fig. 1.

Fig. 1.

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Neuropeptide Y (NPY) induced a sustained increase in spontaneous GABA release from stellate cells in cerebellar slices. A: application of 1 μM NPY increased the frequency of miniature inhibitory postsynaptic currents (mIPSCs) recorded in a cerebellar stellate cell. B: time course of the effect of NPY application on the frequency, amplitude, and decay time constant of mIPSCs in the cell shown in A. Rs, series resistance. C: group data showing that NPY significantly increased mIPSC frequency but not amplitude or decay time (NPY, n = 7; control, n = 9). ANOVA(time), P < 0.05; post hoc test, before vs. after NPY application: *P < 0.05, **P < 0.01, ***P < 0.001.

To investigate further how NPY regulates GABA release from stellate cells, we turned to a cerebellar culture preparation that contains granule cells and stellate/basket cells, which form synapses onto each other (Fiszman et al. 2005). If NPY acts presynaptically to induce an increase in spontaneous GABA release from stellate cells, the effects of NPY should be independent of the target neuron. We would predict that NPY would also enhance mIPSC frequency in cultured granule cells. We recorded mIPSCs in granule cells and found that they were completely blocked by 10 μM bicuculline, a specific GABAA receptor blocker (Fig. 2A). NPY (200 nM) application increased the mIPSC frequency (control, 1.0 ± 0.3 Hz; during NPY application, 1.2 ± 0.4 Hz; n = 9; P < 0.05), which reached ∼50% potentiation after NPY application. The mIPSC frequency remained elevated for at least 30 min (15–30 min after NPY application, 1.5 ± 0.4 Hz; P < 0.01 vs. control; Fig. 2, B–D). Thus NPY induced an increase in spontaneous GABA release from inhibitory neurons [ANOVA test(time), P < 0.05; Fig. 2D]. In this experimental condition, the amplitude [control, 24 ± 3 pA; 15–30 min after NPY application, 27 ± 3 pA; n = 9; ANOVA test(time), P > 0.05; Fig. 2, C and D] and decay time of mIPSCs remained unaltered during and following NPY application [control, 11.0 ± 0.3 ms; 15–30 min after NPY application, 11.6 ± 0.3 ms; n = 9; ANOVA test(time), P > 0.05; Fig. 2, C and D]. These results suggest that NPY triggers a long-lasting increase in spontaneous GABA release from inhibitory interneurons in cerebellar slices and cultures.

Fig. 2.

Fig. 2.

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NPY induced a long-lasting increase in spontaneous GABA release in primary cerebellar cultures. A: application of 10 μM bicuculline blocked mIPSCs recorded in a cerebellar granule cell. B: representative example of the effect of 200 nM NPY on mIPSCs in a granule cell. C: corresponding time course on mIPSC frequency, amplitude, and decay time. D: group data showing that NPY significantly increased mIPSC frequency (NPY, n = 5; control, n = 7). Effect of NPY on mIPSC amplitude and decay time was measured 15–30 min after NPY application. ANOVA(time), P < 0.05; post hoc test, before vs. after NPY application: *P < 0.05.

Expression of Y5r in GABAergic neurons.

To examine the location of the presumptive NPY receptors, we first identified which of the five cloned NPY receptors are expressed in the cerebellum. RT-PCR experiments indicated that mRNAs encoding Y1 and Y5 were present, whereas Y2, Y4, and y6 were not detected (Fig. 3A).

Fig. 3.

Fig. 3.

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Expression of NPY Y5 receptors (Y5rs) in the cerebellum. A: RT-PCR of mouse cerebellum showed the presence of NPY, Y1r and Y5r mRNA. Amplicons of all NPY receptors were detected in whole brain. The control reaction (RT−) contained the Y5 primers but lacked RT. B and C: validation of Y1r and Y5r antibodies (ab). B: summary of the immunoreactivity (-ir) seen in INS-1 cells cotransfected with green fluorescent protein (GFP) and Y1r, Y2r, or Y5r cDNAs and stained with antibodies for Y1r and Y5r. Filled and open circles indicate presence and absence of staining, respectively. C1: Y1r-ir was seen only in cells cotransfected with Y1r and GFP. C2: Y5r-ir was observed exclusively in cells cotransfected with Y5r and GFP. D: cerebellar neurons were double-stained with antibodies for Y5r and vesicular glutamate transporter (vGlut) or vesicular GABA transporter (VGAT). Red (Y5r) and green (vGlut/VGAT) arrows mark processes without colocalization. Yellow arrow highlights a process exhibiting both Y5r-ir and VGAT-ir. D1: few vGlut-ir processes showed Y5r staining. D2: most VGAT-ir processes were also labeled by the Y5r antibody. D3: Y5-ir was typically punctate (left). No staining was found in cerebellar cultures with the Y1r antibody (middle) or in the absence of the primary antibody (right). E: most VGAT-expressing processes were immunoreactive for Y5r, whereas fewer vGlut-positive processes were Y5r-ir-positive (n = 3). #P < 0.05. Scale bars: 10 μm.

We next examined whether Y5rs are expressed in inhibitory interneurons. We tested the specificity of a Y5r antibody on INS-1 cells that were transiently cotransfected with GFP and Y1r, Y2r, or Y5r cDNAs (Fig. 3B). Membrane staining was observed in GFP-positive cells cotransfected with Y5r cDNA but not in cells transfected with Y1r or Y2r cDNAs (Fig. 3, B and C2). We confirmed that Y1rs were expressed in the transfected cells using an Y1r antibody (Fig. 3, B and C1), but we could not obtain specific staining of Y2rs in Y2r-transfected cells using two different commercial Y2r antibodies. These results indicate that Y1 and Y5 mRNA are present in the cerebellum and that the Y5r antibody recognizes Y5rs but not Y1rs.

To determine whether GABAergic neurons in cerebellar cultures express Y5rs, we then stained the cultured cells with antibodies for Y5r and VGAT, the latter being a presynaptic marker for inhibitory synapses. Most of the neuronal processes that were stained for VGAT also had Y5r-ir (81 ± 9%, n = 3; Fig. 3D2, Fig. 3D3, left, and Fig. 3E). In contrast, fewer vGlut-positive processes showed Y5r-ir (33 ± 5%, n = 3; P < 0.05 vs. VGAT-Y5r costaining; Fig. 3, D1 and E). No staining was observed when the primary or secondary antibodies were omitted (Fig. 3D3, right). These results suggest that Y5rs are expressed preferentially on the presynaptic terminals of GABAergic interneurons.

Since Y1r mRNA was also found in the cerebellum (Fig. 3A), we examined the expression of Y1r-ir in the cerebellar cultures. No Y1r-ir was observed (Fig. 3D3, middle).

Blocking Y5rs abolishes the NPY-induced increase in GABA release in cerebellar cultures.

To determine whether activation of Y5rs is required for the NPY-induced increase in GABA release from cerebellar inhibitory interneurons, a Y5r antagonist, L-152,804, was applied 5 min before and during the NPY application. In the presence of L-152,804, NPY failed to enhance the frequency of mIPSCs [15–30 min after NPY + L-152,804, −18 ± 15.5%, n = 4; P < 0.001 vs. NPY alone; ANOVA test(time), P > 0.05; Fig. 4, A–C and E]. Amplitude [control, 33 ± 8 pA; 15–30 min after NPY application, 28 ± 7 pA; n = 4; ANOVA test(time), P > 0.05] and decay time [control, 11.5 ± 0.4 ms; 15–30 min after NPY application, 12.0 ± 0.5 ms; n = 4; ANOVA test(time), P > 0.05] of mIPSCs was not modified by NPY application. A second Y5r antagonist, CGP 71683 (1 μM), also prevented the NPY-induced I-LTP [15–30 min after NPY+CGP 71683, −12.4 ± 4.6%, n = 3; P < 0.05 vs. NPY; ANOVA test(time), P > 0.05; Fig. 4, C–E]. CGP 71683 application did not modify the amplitude [control, 23 ± 3 pA; 15–30 min after NPY application, 22 ± 3 pA; n = 4; ANOVA test(time), P > 0.05] nor decay time [control, 10.8 ± 0.3 ms; 15–30 min after NPY application, 11.3 ± 0.5 ms; n = 4; ANOVA test(time), P > 0.05] of mIPSCs. Neither L-152,804 nor CGP 71683 applied alone altered mIPSC frequency or amplitude [ANOVA test(time), P > 0.05]. DMSO at the concentration used to dissolve the antagonists (0.02%) also did not alter the NPY-induced I-LTP [ANOVA test(time), P < 0.05, interaction 2-way ANOVA test(time × presence of DMSO), P = 0.772; Fig. 4A, top]. These findings suggest that activation of Y5rs is necessary for the long-term enhancement of GABA release that is evoked by NPY.

Fig. 4.

Fig. 4.

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Y5r antagonists prevented the NPY-induced increase in GABA release. A: application of 200 nM NPY induced an increase in mIPSC frequency (top) in cerebellar cultures. In the presence of L-152,804 (1 μM), a specific Y5r antagonist, NPY failed to induce an increase in mIPSC frequency (bottom). B: group data shows that L-152,804 blocked the NPY-induced increase in GABA release (n = 4). ANOVA(time), P < 0.05; post hoc test (before vs. after NPY application): *P < 0.05, **P < 0.01. C: plot of NPY-induced potentiation at 15–30 min after NPY application vs. initial mIPSC frequency shows that the increase in GABA release is independent of initial mIPSC frequency and is blocked by Y5r antagonists L-152,804 and CGP 71683. D: CGP 71683 (1 μM) also prevented the NPY-induced increase in mIPSC frequency. E: summary of the normalized mIPSC frequency 15–30 min after NPY application (CGP 71683; n = 3). Open circles represent individual cells. Interaction 2-way ANOVA(time × treatment), P < 0.05; post hoc test: #P < 0.05, ##P < 0.01.

Activation of Y5rs is necessary and sufficient to trigger the NPY-induced increase in GABA release in cerebellar slices.

We determined the role of Y5rs in the induction of NPY-triggered lasting increase in GABA release from inhibitory interneurons in cerebellar slices. We first examined whether the NPY-induced increase in spontaneous GABA release requires activation of Y5rs. L-152,804 (5 μM) was applied 5–15 min before and during the NPY application. In the presence of L-152,804, NPY failed to enhance the frequency of mIPSCs [15–30 min after NPY + L-152,804, −1 ± 11%, n = 4; paired t-test, P < 0.05 vs. NPY alone; ANOVA test(time), P > 0.05; interaction 2-way ANOVA test(time × presence of Y5r antagonist), P < 0.001; Fig. 5, A and C]. L-152,804 did not alter the amplitude [control, 136 ± 22 pA; 15–30 min after NPY application, 106 ± 22 pA; n = 4; ANOVA test(time), P > 0.05] and decay time [control, 5.6 ± 0.6 ms; 15–30 min after NPY application, 7.3 ± 1 ms; n = 4; ANOVA test(time), P < 0.05] of mIPSCs. Thus activation of Y5rs is necessary for the enhancement of GABA release that is induced by NPY.

Fig. 5.

Fig. 5.

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Y5rs, but not Y1rs or Y2rs, mediated the NPY-induced increase in spontaneous GABA release in cerebellar slices. A: coapplication of L-152,804 with NPY blocked the NPY-induced increase in GABA release. B: coapplication of BIBP3236 (BIBP) and BIIE-0246 (BIIE) with NPY did not prevent the NPY-induced increase in GABA release. C: group data showing that the effect of NPY on mIPSC frequency was prevented in the presence of L-152,804 [ANOVA(time), P > 0.05; n = 4] but not in the presence of BIBP3236 and BIIE-0246 [ANOVA(time), P < 0.05; post hoc test(before vs. after NPY application): *P < 0.05; n = 4]. D: representative example showing that application of 400 nM [cPP1–7,NPY19–23,Ala31,Aib32,Gln34]-hPancreatic Polypeptide (a Y5 agonist) induced an increase in mIPSC frequency. The time course of the effect is shown on the right. E and F: group data showing that activation of Y5rs with [cPP1–7,NPY19–23,Ala31,Aib32,Gln34]-hPancreatic Polypeptide led to a significant increase in mIPSC frequency (Y5r agonist, n = 3; control, n = 5). ANOVA(time), P < 0.05; post hoc test: before vs. after Y5r agonist application, *P < 0.05, ###P < 0.001.

To test any possible involvement of Y1rs and Y2rs in the NPY-induced increase of GABA release, we coapplied specific antagonists for Y1r, 1 μM BIBP3236, and Y2r, 1 μM BIIE-0246, 15 min before and during NPY application. Following blockade of Y1rs and Y2rs, application of NPY was still able to induce an increase in mIPSC frequency [15–30 min after NPY + BIBP3236 + BIIE-0246, 33 ± 7%, n = 4; paired t-test, P > 0.05 vs. NPY alone; ANOVA test(time), P < 0.001; interaction 2-way ANOVA test(time × presence of Y1/2r antagonist), P > 0.05; Fig. 5, B and C] but did not affect the amplitude [control, 155 ± 54 pA; 15–30 min after NPY application, 160 ± 57 pA; n = 4; ANOVA test(time), P > 0.05] or decay time [control, 6.3 ± 0.8 ms; 15–30 min after NPY application, 6.5 ± 0.9 ms; n = 4; ANOVA test(time), P < 0.05]. Therefore, activation of Y1rs or Y2rs is not necessary for the NPY-dependent increase in GABA release.

We next examined whether activation of Y5rs by a Y5 agonist was sufficient to enhance GABA release. Application of 400 nM [cPP1–7,NPY19–23,Ala31,Aib32,Gln34]-hPancreatic Polypeptide, a specific Y5r agonist (Cabrele et al. 2000), for 15 min induced a sustained increase in mIPSC frequency [control, 0.4 ± 0.1 Hz; 15–30 min after Y5r agonist application, 0.6 ± 0.1 Hz; n = 3, ANOVA test(time), P < 0.05; Fig. 5, D–F] without changing the amplitude [control, 199 ± 51 pA; 15–30 min after NPY application, 161 ± 53 pA; n = 3; ANOVA test(time), P > 0.05] or decay time [control, 7.6 ± 0.5 ms; 15–30 min after NPY application, 9.4 ± 0.5 ms; n = 3; ANOVA test(time), P > 0.1] of mIPSCs. The Y5r agonist-induced potentiation of mIPSC frequency [44 ± 7%; interaction 2-way ANOVA test(time × NPY/Y5r agonist), P > 0.05; Fig. 5, E and F] mimicked the NPY-induced increase in spontaneous GABA release. These data suggest that activation of Y5r is not only necessary, but also sufficient to induce a lasting increase in GABA release from inhibitory interneurons.

NPY-induced increase in GABA release is a form of long-term plasticity.

Our results so far show that NPY acting via Y5rs can induce an increase in GABA release that is long-lasting. If activation of Y5rs during NPY application triggers a lasting increase in spontaneous GABA release, the potentiation once induced should be independent of Y5rs. To test this idea, we applied NPY to induce an increase in mIPSC frequency, and, after it had reached a plateau value (15 min of stable potentiation), we applied the Y5r antagonist L-152,804 (1 μM) for at least 10 min. NPY application induced a sustained increase in mIPSC frequency (plateau value: 63 ± 14% potentiation, n = 4; paired t-test, P < 0.01; Fig. 6, A and B). The Y5r antagonist failed to attenuate the NPY-induced increase in GABA release, and the NPY-induced potentiation in mIPSC frequency remained unaltered (L-152,804, 65 ± 15% potentiation, n = 4; paired t-test, P > 0.05). Furthermore, increasing the concentration of L-152,804 to 5 μM also did not reverse the increase of mIPSC frequency induced by NPY (∼50% potentiation; n = 2; Fig. 6C). These results suggest that the NPY-induced lasting increase of GABA release from inhibitory interneurons is a form of LTP.

Fig. 6.

Fig. 6.

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The NPY-induced increase in GABA release is a form of long-term potentiation of inhibitory synapses (I-LTP). A: representative example showing that the application of L-152,804 (1 μM) 30 min after washout of NPY did not alter mIPSC frequency. B: time course of the experiment shown in A indicates that once the NPY-induced increase in mIPSC frequency was initiated, it was not reversed by the application of a Y5r antagonist. C: group data showing that L-152,804 (1 μM, during the last 8 min of L-152,804 application) did not attenuate the NPY-induced LTP (n = 4). In 2 cells, the L-152,804 concentration was subsequently raised to 5 μM, but this did not affect the I-LTP.

DISCUSSION

NPY has emerged as an important regulator of GABA release from inhibitory interneurons in the central nervous system. Although the activation of Y1rs and Y2rs is known to reduce membrane excitability and suppress neurotransmitter release, Y5rs are also expressed in many GABAergic neurons. We found that cerebellar inhibitory interneurons express presynaptic Y5rs and investigated whether activation of these receptors could modulate inhibitory transmission. In contrast to the NPY-induced transient suppression of GABA release observed at a number of synapses, NPY triggered a long-lasting increase in spontaneous GABA release from cerebellar GABAergic interneurons. Inhibition of Y5rs, but not of Y1rs or Y2rs, completely abolished the enhancement of GABA secretion induced by NPY, and the selective activation of Y5r was able to trigger this phenomenon. Thus NPY acts on presynaptic Y5rs to induce a sustained increase in GABA release.

As a neuromodulator, NPY is known to reduce GABA release transiently onto neurons of the thalamus, hypothalamus, hippocampus, and spinal cord (Chen and van den Pol 1996; Kash and Winder 2006; Moran et al. 2004; Sperk et al. 2007), often by modulating calcium (Acuna-Goycolea et al. 2005; Alvaro et al. 2009; Bleakman et al. 1991; Ewald et al. 1988; Fu et al. 2004; Obrietan and van den Pol 1996; Qian et al. 1997; Silva et al. 2003; Sun et al. 2001b; Toth et al. 1993) and inwardly rectifying potassium channels (Acuna-Goycolea et al. 2005; Fu et al. 2004; Sun et al. 2001a,b, 2003; Zidichouski et al. 1990). In neocortical neurons, NPY induces a long-lasting increase in calcium-dependent GABA release, presumably by enhancing the excitability of presynaptic inhibitory neurons (Bacci et al. 2002). Our results show that NPY can trigger a sustained increase in spontaneous GABA release in cerebellar neurons. Although transient suppression of GABA release by NPY is often mediated by presynaptic Y2rs (Bleakman et al. 1991), we found that the induction of I-LTP requires the activation of Y5rs that are present at many GABAergic terminals. Since Y5rs have been implicated in the inhibition of seizure activity and in controlling anxiety (Marsh et al. 1999; Woldbye et al. 2005), their presynaptic localization suggests a new role for Y5r in the modulation of GABA release. An NPY-dependent enhancement of GABA release via Y5rs may serve as a possible mechanism for its known antiepileptic and anxiolytic properties.

In this study, three pieces of evidence suggested that Y5rs are expressed in cerebellar GABAergic neurons. First, Y5r mRNA was detected in the cerebellum using RT-PCR. Second, Y5r-ir was found in the processes of cultured interneurons expressing VGAT. Third, the use of pharmacological tools allowed us to show that activation of Y5rs was necessary and sufficient to induce an I-LTP. These results support the notion that functional Y5rs are present in the presynaptic terminals of GABAergic interneurons, in agreement with previous reports of moderate levels of Y5r-ir in the cerebellum and the absence of Y5r mRNA in Purkinje and granule cells (Akiyama et al. 2008; Morin and Gehlert 2006). However, our observation of Y5r-ir in a fraction of vGlut-ir processes (Fig. 3) suggests that some excitatory neurons also express Y5rs, and thus the presence of Y5rs in cerebellar granule cells needs further investigation. Our results are consistent with previous observations that Y2r mRNA is only transiently expressed in immature granule cells of developing cerebellum and is absent in the adult (Neveu et al. 2002). Several studies have shown the expression of Y1r mRNA and Y1r-ir in Purkinje cells (Kopp et al. 2002; Wolak et al. 2003). We also detected the expression of Y1r mRNA in the cerebellum. However, we did not find any Y1r-ir in the cerebellar cultures because Purkinje cells do not survive under these culture conditions. Consistently, blockade of these receptors together with Y2rs did not affect the NPY-induced I-LTP.

Could the NPY-induced I-LTP contribute to cerebellar physiology? NPY is expressed in both climbing and mossy fibers (Laemle et al. 1991; Ueyama et al. 1994), and thereby endogenous NPY is likely to be released by stimulation of these two major inputs to the cerebellum. Recent studies have suggested that the cerebellum contains a circadian oscillator involved in food anticipation (Mendoza et al. 2010) and that NPY gene transcription in the cerebellum is modulated by food deprivation (Liu et al. 2008). Thus the NPY-induced enhancement of GABA release could play a role in the modulation of feeding behavior. The cerebellum is also involved in several forms of associative learning, and interneuronal inhibition onto Purkinje cells is enhanced in the association process during fear conditioning (Scelfo et al. 2008). We have recently shown that increased GABA release from stellate cells can induce burst-firing activity in inhibitory interneurons (Lachamp et al. 2009). Thus an NPY-induced GABA release from inhibitory interneurons may also contribute to the fear-induced GABA release in the cerebellum and so modulate cerebellar network activity.

GRANTS

This work was supported by National Science Foundation Grant IBN-0964517 (S. J. Liu) and National Institutes of Health Grants NS-58867 (S. J. Liu) and DK-080441 (M. D. Whim).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

C.J.D., M.D.W., and S.J.L. conception and design of research; C.J.D., P.R., and M.D.W. performed experiments; C.J.D., P.R., and M.D.W. analyzed data; C.J.D., M.D.W., and S.J.L. interpreted results of experiments; C.J.D. and M.D.W. prepared figures; C.J.D. and S.J.L. drafted manuscript; C.J.D., M.D.W., and S.J.L. edited and revised manuscript; C.J.D., P.R., M.D.W., and S.J.L. approved final version of manuscript.

ACKNOWLEDGMENTS

We thank Eric Lazartigues, Charles Nichols, Iaroslav Savtchouk, and Philippe Lachamp for experimental advice and helpful discussions.

Present address of P. Ramamoorthy: Dept. of Molecular and Integrative Physiology, Univ. of Kansas Medical Center, Kansas City, KS 66160.

REFERENCES

  1. Acuna-Goycolea C, Tamamaki N, Yanagawa Y, Obata K, van den Pol AN. Mechanisms of neuropeptide Y, peptide YY, and pancreatic polypeptide inhibition of identified green fluorescent protein-expressing GABA neurons in the hypothalamic neuroendocrine arcuate nucleus. J Neurosci 25: 7406–7419, 2005 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Akiyama K, Nakanishi S, Nakamura NH, Naito T. Gene expression profiling of neuropeptides in mouse cerebellum, hippocampus, and retina. Nutrition 24: 918–923, 2008 [DOI] [PubMed] [Google Scholar]
  3. Alvaro AR, Rosmaninho-Salgado J, Ambrosio AF, Cavadas C. Neuropeptide Y inhibits [Ca2+]i changes in rat retinal neurons through NPY Y1, Y4, and Y5 receptors. J Neurochem 109: 1508–1515, 2009 [DOI] [PubMed] [Google Scholar]
  4. Bacci A, Huguenard JR, Prince DA. Differential modulation of synaptic transmission by neuropeptide Y in rat neocortical neurons. Proc Natl Acad Sci USA 99: 17125–17130, 2002 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bleakman D, Colmers WF, Fournier A, Miller RJ. Neuropeptide Y inhibits Ca2+ influx into cultured dorsal root ganglion neurones of the rat via a Y2 receptor. Br J Pharmacol 103: 1781–1789, 1991 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cabrele C, Langer M, Bader R, Wieland HA, Doods HN, Zerbe O, Beck-Sickinger AG. The first selective agonist for the neuropeptide YY5 receptor increases food intake in rats. J Biol Chem 275: 36043–36048, 2000 [DOI] [PubMed] [Google Scholar]
  7. Campbell RE, ffrench-Mullen JM, Cowley MA, Smith MS, Grove KL. Hypothalamic circuitry of neuropeptide Y regulation of neuroendocrine function and food intake via the Y5 receptor subtype. Neuroendocrinology 74: 106–119, 2001 [DOI] [PubMed] [Google Scholar]
  8. Chee MJ, Colmers WF. Y eat? Nutrition 24: 869–877, 2008 [DOI] [PubMed] [Google Scholar]
  9. Chen G, van den Pol AN. Multiple NPY receptors coexist in pre- and postsynaptic sites: inhibition of GABA release in isolated self-innervating SCN neurons. J Neurosci 16: 7711–7724, 1996 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ewald DA, Matthies HJ, Perney TM, Walker MW, Miller RJ. The effect of down regulation of protein kinase C on the inhibitory modulation of dorsal root ganglion neuron Ca2+ currents by neuropeptide Y. J Neurosci 8: 2447–2451, 1988 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fiszman ML, Barberis A, Lu C, Fu Z, Erdelyi F, Szabo G, Vicini S. NMDA receptors increase the size of GABAergic terminals and enhance GABA release. J Neurosci 25: 2024–2031, 2005 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fu LY, Acuna-Goycolea C, van den Pol AN. Neuropeptide Y inhibits hypocretin/orexin neurons by multiple presynaptic and postsynaptic mechanisms: tonic depression of the hypothalamic arousal system. J Neurosci 24: 8741–8751, 2004 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Grove KL, Campbell RE, Ffrench-Mullen JM, Cowley MA, Smith MS. Neuropeptide Y Y5 receptor protein in the cortical/limbic system and brainstem of the rat: expression on gamma-aminobutyric acid and corticotropin-releasing hormone neurons. Neuroscience 100: 731–740, 2000 [DOI] [PubMed] [Google Scholar]
  14. Guo H, Castro PA, Palmiter RD, Baraban SC. Y5 receptors mediate neuropeptide Y actions at excitatory synapses in area CA3 of the mouse hippocampus. J Neurophysiol 87: 558–566, 2002 [DOI] [PubMed] [Google Scholar]
  15. Karlsson RM, Holmes A, Heilig M, Crawley JN. Anxiolytic-like actions of centrally-administered neuropeptide Y, but not galanin, in C57BL/6J mice. Pharmacol Biochem Behav 80: 427–436, 2005 [DOI] [PubMed] [Google Scholar]
  16. Kash TL, Winder DG. Neuropeptide Y and corticotropin-releasing factor bi-directionally modulate inhibitory synaptic transmission in the bed nucleus of the stria terminalis. Neuropharmacology 51: 1013–1022, 2006 [DOI] [PubMed] [Google Scholar]
  17. Klenke U, Constantin S, Wray S. Neuropeptide Y directly inhibits neuronal activity in a subpopulation of gonadotropin-releasing hormone-1 neurons via Y1 receptors. Endocrinology 151: 2736–2746, 2010 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kopp J, Xu ZQ, Zhang X, Pedrazzini T, Herzog H, Kresse A, Wong H, Walsh JH, Hokfelt T. Expression of the neuropeptide Y Y1 receptor in the CNS of rat and of wild-type and Y1 receptor knock-out mice. Focus on immunohistochemical localization. Neuroscience 111: 443–532, 2002 [DOI] [PubMed] [Google Scholar]
  19. Lachamp PM, Liu Y, Liu SJ. Glutamatergic modulation of cerebellar interneuron activity is mediated by an enhancement of GABA release and requires protein kinase A/RIM1alpha signaling. J Neurosci 29: 381–392, 2009 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Laemle LK, Dlugos CA, Cotter JR. Neuropeptide Y innervation of the cerebellum in the little brown bat (Myotis lucifugus). Neurosci Lett 122: 25–28, 1991 [DOI] [PubMed] [Google Scholar]
  21. Liu HZ, Li XY, Tong JJ, Qiu ZY, Zhan HC, Sha JN, Peng KM. Duck cerebellum participates in regulation of food intake via the neurotransmitters serotonin and neuropeptide Y. Nutr Neurosci 11: 200–206, 2008 [DOI] [PubMed] [Google Scholar]
  22. Liu SQ, Cull-Candy SG. Synaptic activity at calcium-permeable AMPA receptors induces a switch in receptor subtype. Nature 405: 454–458, 2000 [DOI] [PubMed] [Google Scholar]
  23. Marsh DJ, Baraban SC, Hollopeter G, Palmiter RD. Role of the Y5 neuropeptide Y receptor in limbic seizures. Proc Natl Acad Sci USA 96: 13518–13523, 1999 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mendoza J, Pevet P, Felder-Schmittbuhl MP, Bailly Y, Challet E. The cerebellum harbors a circadian oscillator involved in food anticipation. J Neurosci 30: 1894–1904, 2010 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mitchell GC, Wang Q, Ramamoorthy P, Whim MD. A common single nucleotide polymorphism alters the synthesis and secretion of neuropeptide Y. J Neurosci 28: 14428–14434, 2008 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Morales-Medina JC, Dumont Y, Quirion R. A possible role of neuropeptide Y in depression and stress. Brain Res 1314: 194–205, 2010 [DOI] [PubMed] [Google Scholar]
  27. Moran TD, Colmers WF, Smith PA. Opioid-like actions of neuropeptide Y in rat substantia gelatinosa: Y1 suppression of inhibition and Y2 suppression of excitation. J Neurophysiol 92: 3266–3275, 2004 [DOI] [PubMed] [Google Scholar]
  28. Morin SM, Gehlert DR. Distribution of NPY Y5-like immunoreactivity in the rat brain. J Mol Neurosci 29: 109–114, 2006 [DOI] [PubMed] [Google Scholar]
  29. Neveu I, Remy S, Naveilhan P. The neuropeptide Y receptors, Y1 and Y2, are transiently and differentially expressed in the developing cerebellum. Neuroscience 113: 767–777, 2002 [DOI] [PubMed] [Google Scholar]
  30. Obrietan K, van den Pol AN. Neuropeptide Y depresses GABA-mediated calcium transients in developing suprachiasmatic nucleus neurons: a novel form of calcium long-term depression. J Neurosci 16: 3521–3533, 1996 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Qian J, Colmers WF, Saggau P. Inhibition of synaptic transmission by neuropeptide Y in rat hippocampal area CA1: modulation of presynaptic Ca2+ entry. J Neurosci 17: 8169–8177, 1997 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ramamoorthy P, Wang Q, Whim MD. Cell type-dependent trafficking of neuropeptide Y-containing dense core granules in CNS neurons. J Neurosci 31: 14783–14788, 2011 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ramamoorthy P, Whim MD. Trafficking and fusion of neuropeptide Y-containing dense-core granules in astrocytes. J Neurosci 28: 13815–13827, 2008 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Scelfo B, Sacchetti B, Strata P. Learning-related long-term potentiation of inhibitory synapses in the cerebellar cortex. Proc Natl Acad Sci USA 105: 769–774, 2008 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Shinoda H, Marini AM, Cosi C, Schwartz JP. Brain region and gene specificity of neuropeptide gene expression in cultured astrocytes. Science 245: 415–417, 1989 [DOI] [PubMed] [Google Scholar]
  36. Silva AP, Carvalho AP, Carvalho CM, Malva JO. Functional interaction between neuropeptide Y receptors and modulation of calcium channels in the rat hippocampus. Neuropharmacology 44: 282–292, 2003 [DOI] [PubMed] [Google Scholar]
  37. Sorensen G, Lindberg C, Wortwein G, Bolwig TG, Woldbye DP. Differential roles for neuropeptide Y Y1 and Y5 receptors in anxiety and sedation. J Neurosci Res 77: 723–729, 2004 [DOI] [PubMed] [Google Scholar]
  38. Sperk G, Hamilton T, Colmers WF. Neuropeptide Y in the dentate gyrus. Prog Brain Res 163: 285–297, 2007 [DOI] [PubMed] [Google Scholar]
  39. Sun QQ, Akk G, Huguenard JR, Prince DA. Differential regulation of GABA release and neuronal excitability mediated by neuropeptide Y1 and Y2 receptors in rat thalamic neurons. J Physiol 531: 81–94, 2001a [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sun QQ, Baraban SC, Prince DA, Huguenard JR. Target-specific neuropeptide Y-ergic synaptic inhibition and its network consequences within the mammalian thalamus. J Neurosci 23: 9639–9649, 2003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sun QQ, Huguenard JR, Prince DA. Neuropeptide Y receptors differentially modulate G-protein-activated inwardly rectifying K+ channels and high-voltage-activated Ca2+ channels in rat thalamic neurons. J Physiol 531: 67–79, 2001b [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Toth PT, Bindokas VP, Bleakman D, Colmers WF, Miller RJ. Mechanism of presynaptic inhibition by neuropeptide Y at sympathetic nerve terminals. Nature 364: 635–639, 1993 [DOI] [PubMed] [Google Scholar]
  43. Ubink R, Calza L, Hokfelt T. ‘Neuro’-peptides in glia: focus on NPY and galanin. Trends Neurosci 26: 604–609, 2003 [DOI] [PubMed] [Google Scholar]
  44. Ueyama T, Houtani T, Nakagawa H, Baba K, Ikeda M, Yamashita T, Sugimoto T. A subpopulation of olivocerebellar projection neurons express neuropeptide Y. Brain Res 634: 353–357, 1994 [DOI] [PubMed] [Google Scholar]
  45. Whim MD. Pancreatic beta cells synthesize neuropeptide Y and can rapidly release peptide co-transmitters. PLoS One 6: e19478, 2011 [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wolak ML, DeJoseph MR, Cator AD, Mokashi AS, Brownfield MS, Urban JH. Comparative distribution of neuropeptide Y Y1 and Y5 receptors in the rat brain by using immunohistochemistry. J Comp Neurol 464: 285–311, 2003 [DOI] [PubMed] [Google Scholar]
  47. Woldbye DP, Nanobashvili A, Sorensen AT, Husum H, Bolwig TG, Sorensen G, Ernfors P, Kokaia M. Differential suppression of seizures via Y2 and Y5 neuropeptide Y receptors. Neurobiol Dis 20: 760–772, 2005 [DOI] [PubMed] [Google Scholar]
  48. Wulff P, Schonewille M, Renzi M, Viltono L, Sassoe-Pognetto M, Badura A, Gao Z, Hoebeek FE, van Dorp S, Wisden W, Farrant M, De Zeeuw CI. Synaptic inhibition of Purkinje cells mediates consolidation of vestibulo-cerebellar motor learning. Nat Neurosci 12: 1042–1049, 2009 [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Zidichouski JA, Chen H, Smith PA. Neuropeptide Y activates inwardly-rectifying K(+)-channels in C-cells of amphibian sympathetic ganglia. Neurosci Lett 117: 123–128, 1990 [DOI] [PubMed] [Google Scholar]

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