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Endocrinology Vol. 143, No. 2 535-543
Copyright © 2002 by The Endocrine Society

NEUROENDOCRINOLOGY

Multiple NPY Receptors Inhibit GABAA Synaptic Responses of Rat Medial Parvocellular Effector Neurons in the Hypothalamic Paraventricular Nucleus

Nina Pronchuk, Annette G. Beck-Sickinger and William F. Colmers

Department of Pharmacology (N.P., W.F.C.), University of Alberta, Edmonton, Alberta, Canada T6G 2H7; and Institute of Biochemistry (A.G.B.-S.), University of Leipzig, Leipzig D-04103, Germany

Address all correspondence and requests for reprints to: Dr. William F. Colmers, Department of Pharmacology, University of Alberta, 9-36 Medical Sciences Building, Edmonton, Alberta, Canada T6G 2H7. E-mail: william.colmers{at}ualberta.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have recently shown that NPY and {alpha}-melanocyte-stimulating hormone, which potently induce or inhibit feeding, respectively, have opposing modulatory actions on GABAergic synapses in the medial parvocellular region of the paraventricular hypothalamic nucleus (mpPVN). Because this action might underlie the effects of NPY on feeding, we have examined the pharmacology of NPY responses using electrophysiological recordings.

Focal electrical stimulation within the PVN elicited a GABAA synaptic response in some mpPVN neurons, which was reversibly inhibited by NPY in a concentration-dependent manner (EC50 = 28 nM). NPY did not alter the response to the GABAA agonist, muscimol.

Agonist responses to NPY analogs were not consistent with a single NPY receptor subtype; the most subtype selective agonists were less effective than the more broadly selective ones. Antagonist blockade of individual receptor subtypes partly inhibited NPY action, while fully blocking effects of selective agonists. Combining Y1 and Y5 antagonists blocked actions of NPY entirely, but the Y2 antagonist also completely blocked actions of NPY in some neurons.

NPY inhibits GABAA synaptic transmission onto mpPVN neurons, but this can be mediated by three different NPY receptors. Controversy regarding the receptor or receptor subtypes involved in NPY-mediated feeding may arise from the multiple NPY receptors present.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EXOGENOUS NPY IS one of the most potent orexigens known (1) and has been suggested to be a physiological regulator of food intake (2, 3). Feeding responses can be elicited with NPY microinjection into several hypothalamic areas, including the perifornical hypothalamus and the paraventricular nucleus (PVN,4). NPY projections are highly concentrated in the PVN (5, 6) and arise mainly from a population of arcuate nucleus (ARC) neurons, and from other areas, especially the brain stem (7, 8, 9, 10). The NPY projections also contain agouti gene-related peptide (AGRP), another potent orexigen that acts at least in part by blocking melanocortin-4 receptors (6, 11).

The identity of the receptor(s) that mediate the orexigenic effects of NPY is controversial. NPY receptors can be pharmacologically distinguished in part by their responses to the related, naturally occurring agonist peptides, peptide YY (PYY) and pancreatic polypeptide (PP), and to C-terminal peptide fragments and analogs (12, 13). The receptor subtype(s) involved in the NPY feeding response was initially described as "Y1-like," because agonists, which demonstrate some selectivity for this receptor, can induce feeding (14). Y1 antagonists have been reported to significantly inhibit NPY-induced feeding (15, 16). However, the high orexigenic potency of NPY2–36 and NPY 3–36, but not NPY13–36, all of which generally prefer Y2 receptors (15, 17) is inconsistent with a Y1 receptor action. Furthermore, knockout of the Y1 receptor did not alter food intake in mice (18, 19).

The Y5 receptor has also been implicated in the feeding induced by NPY (20). This receptor is not blocked by Y1 antagonists (21, 22) but does respond to nearly all agonists known to activate the Y1 receptor, in addition to NPY2–36, NPY3–36 and human PP (hPP). It has been proposed that the Y5 receptor alone may mediate the orexigenic effect of NPY (20, 21), although knockout of the mouse Y5 receptor also did not alter food intake (23).

To examine hypothalamic mechanisms regulating feeding, we recently developed a rat hypothalamic slice preparation in which we identified neuronal responses sensitive to energy balance-related signals. Specifically, in medial parvocellular PVN (mpPVN) neurons, NPY reduced a GABAA-mediated inhibitory postsynaptic current (IPSC,24, 25). In the same neurons, this IPSC was increased by anorectic agonists of the melanocortin-4 (MC4) receptor. AGRP, a natural antagonist of MC-4 receptors blocked melanocortin effects on this synapse (6). These responses suggest that this population of GABA terminals might act to integrate positive and negative signals related to energy balance. If so, the pharmacological characteristics of the NPY receptors mediating this action should be relevant to the mechanism by which NPY regulates energy balance.

Therefore, we have studied the pharmacological characteristics of the NPY-mediated response in the mpPVN in detail, using the in vitro hypothalamic slice preparation. NPY inhibits the IPSC in mpPVN cells in a concentration-dependent manner and does not affect postsynaptic properties or the response to GABAA receptor agonist application. Inhibition by NPY of the IPSC was reduced, but not abolished by either Y1 or Y5 antagonists, although coapplication of a Y1 and a Y5 receptor antagonist did block the effect of NPY. Unexpectedly, a Y2 antagonist always reduced, or abolished the response to NPY. Multiple receptors mediate the response to NPY in mpPVN, consistent with the unusual pharmacology described by others for the NPY feeding response in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of brain slices
Hypothalamic slices were prepared from brains of male Sprague Dawley rats (21–35 d old). Rats were killed according to a protocol approved by the University of Alberta Health Sciences Laboratory Animal Welfare Committee. The brain was rapidly removed and placed in ice-cold (<4 C) artificial cerebrospinal fluid (aCSF) slicing solution containing (in mM) 118 NaCl, 3 KCl, 6 MgSO4, 2 Ca2+, 1.4 NaH2PO4, 26 NaHCO3, and 10 glucose and saturated with 95% O2- 5% CO2 (carbogen). A block of tissue containing the hypothalamus was dissected and cut into coronal slices (400 µm) with a vibratome (Technical Products, Inc., St. Louis, MO). Slices of hypothalamus containing PVN were obtained just caudal to the optic chiasm. Slices were maintained in carbogenated bath perfusion aCSF, containing (in mM) 124 NaCl, 3 KCl, 1.3 MgSO4, 2.5 Ca2+, 1.4 NaH2PO4, 26 NaHCO3, and 10 glucose at 32 C for at least 1 h before recording. This aCSF was used in all subsequent parts of an experiment.

Electrophysiological recording
Single hypothalamic slices were transferred into a recording chamber and perfused, submerged in bath perfusion aCSF at 34 ± 0.5 C. Recordings were made from neurons in the medial parvocellular area of the PVN using the "blind" whole cell patch clamping technique (26, 27). Glass electrodes were pulled on a two-stage puller (PP-83, Narishige) from borosilicate glass (World Precision Instruments, Sarasota, FL) and had initial tip resistances of 5–6 M{Omega} in the bath when filled with an internal solution of the following composition (in mM): 135 potassium gluconate, 2 KCl, 5 HEPES, 5 MgATP, 0.3 NaGTP, 5 EGTA, 5 creatine phosphate, 0.1 BAPTA, pH adjusted to 7.25 with KOH, osmolarity 295–298 mOsm. We routinely included neurobiotin (0.15%; Vector Laboratories, Inc., Burlingame, CA) or biocytin (0.15%; Sigma, St. Louis, MO) in the pipette solution for subsequent identification of the neurons. Electrodes were connected to the headstage of an Axoclamp 2A amplifier (Axon Instruments, Burlingame, CA) used either in the bridge current clamp or continuous single electrode voltage clamp mode.

Medial parvocellular neurons were identified by their position in the slice (Fig. 1Go) and by their electrophysiological properties. Neurons we chose for this study generally had properties similar to the nonbursting, low-threshold spike neurons described by Hoffman et al. (28), although we did not classify them further in this study. Parvocellular neurons could be readily differentiated from magnocellular neurons on the basis of action potential waveform and amplitude, resting potential and input resistance. These cells normally rested between -45 and -55 mV, their input resistance, estimated from their slope conductance between about -90 and -60 mV, was 295 ± 13 M{Omega} (n = 6), and they showed no sign of bursting behavior. Neurons were routinely held in voltage clamp at -60 mV. Neurons were only studied if their holding current and access resistance remained stable in voltage clamp for 10–15 min before any other manipulations.



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  		Figure 1. Diagram showing the location of the neurons tested in parvocellular part of medial paraventricular nucleus. Each circle represents an individual neuron from which the recording was successful. 3V, Third ventricle; M, magnocellular region of PVN; F, fornix. Scale bar, 0.5 mm.

 
Synaptic potentials were recorded upon electrical stimulation via a sharpened, monopolar tungsten electrode, which was positioned in the PVN after the neuron was acquired, and repositioned within this region as necessary to elicit optimal synaptic responses. Neurons were held at or near -40 mV during synaptic stimulation, where the GABAA response is an outward (positive) current, then returned to -60 mV once synaptic responses were acquired. Pairs of stimuli (10–20 V, 100–200 µsec, 80 msec inter stimulus interval) generally elicited both inward and outward synaptic currents at the holding potential. Outward currents were sensitive to bicuculline (10 µM) and picrotoxin (50 µM), indicating they were mediated by GABA acting at GABAA receptors. Inward synaptic currents could be blocked by 6-nitro-7-sulfamoylbenzo (f)quinoxaline-2,3-dione (NBQX, 1 µM, gift of Novo Nordisk, Maløov, Denmark) and DL-2-amino-5-phosphonovaleric acid (50 µM) and therefore were mediated by glutamate acting at {alpha}-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate receptors. At least three control records were taken at 5-min intervals to ensure the stability of synaptic responses before drug application. Each synaptic recording was the digital average of three successive responses elicited at 10-sec intervals. To ensure the stability of recordings, and determine if changes occurred to subthreshold postsynaptic conductances, the membrane current response to voltage ramps (swept from -110 to -40 mV over 2 sec) was assessed just before drug application and during the peak drug effect. In some cases, a train of hyperpolarizing voltage steps (10 mV, 20 msec duration) was superimposed on the voltage ramp to assess changes in membrane conductance throughout the voltage range tested.

All agonists and antagonists were dissolved in warmed, carbogenated aCSF just before use and applied via bath perfusion. Peptides were kept at -20 C as concentrated aliquots until immediately before use. Porcine NPY (pNPY) was purchased from Dr. S. St.-Pierre (Peptidec Technologies, Montréal, Québec, Canada); pPYY, pNPY 2–36, rat and human PP and D-Trp32NPY were purchased from Bachem (Torrance, CA). BIBP3226, a Y1 receptor-selective antagonist (29), was purchased from Peninsula Laboratories, Inc. (Belmont, CA). The Y2-selective antagonist, BIIE 0246 (30) was a gift of Dr. Henri Doods, Boehringer Ingelheim GmbH (Biberach, Germany). The Y5 antagonists, Novartis1 (trans-2-nitrobenzene-2-sulfonic acid (4-(2-naphthylmethylamino) methyl) cyclohexyl methyl) amide) and Novartis2 (trans-naphthalene-2-sulfonic acid 4-((4-(2-dimethylaminopropyl amino) quinazolin-2-ylamino) methyl) cyclohexylmethyl) amide), were a gift of Dr. P. Hipskind, Lilly Research Laboratories. All other peptides were synthesized by solid phase peptide synthesis as described previously (31, 32, 33). All chemicals for aCSF were obtained from BDH (Toronto, Ontario, Canada) and all other chemicals were obtained from Sigma.

Histological procedures
The procedure, with some modifications, was similar to the procedure described elsewhere (34). Briefly, after the recordings, slices with biocytin- or neurobiotin-filled neurons were fixed for 12–24 h at 4 C in 100 mM PBS (pH 7.4), containing 4% paraformaldehyde. Then the slices were washed (10 min x 3) in PBS, incubated in 2% Triton X-100 for 1 h, and next in avidin-biotinylated horseradish peroxidase (ABC Elite Kit, Vector Laboratories, Inc.) for 2 h. The cells were visualized by treatment with diaminobenzidine (0.05%).

Statistical analysis
The effects of drugs on the IPSC are expressed as percent inhibition of the control responses obtained immediately before application of the test substance. The magnitudes of agonist responses were calculated only from cells demonstrating measurable (>10%) peak agonist action with substantial (>50%) reversal upon agonist washout. Data are expressed as means ± SEM. Wherever possible, paired t test comparisons were performed, both for the significance of an agonist effect and for comparison of an agonist’s action in the absence or presence of an antagonist. The effects of different agonists at a given concentration were compared using t tests for population means.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
NPY and related peptides reduce inhibitory postsynaptic currents
Whole-cell patch clamp recordings were routinely made from medial parvocellular neurons of the PVN. In a large subpopulation of these neurons, focal electrical stimulation in the PVN elicited mostly IPSCs. Such IPSCs reversed at between -60 to -65 mV, and, when tested, were always sensitive to the GABAA receptor antagonists picrotoxin or bicuculline (n = 6, Fig. 2AGo). In some neurons, both IPSCs and glutamate-mediated excitatory postsynaptic currents could be observed. In some experiments with neurons exhibiting such mixed responses, we pharmacologically isolated the IPSC by superfusing the slices for 10 min with NBQX (1 µM) and DL-2-amino-5-phosphonovaleric acid (50 µM) to block AMPA and N-methyl-D-aspartate receptors, respectively. This treatment had a small but measurable effect on the synaptic current (Fig. 2BGo). However, in most experiments, synaptic currents were elicited without the addition of glutamate blockers, whereas the cell was held positive to rest at -40 mV. At this potential, the IPSC was an outward current, and the excitatory postsynaptic current was a relatively small inward current.



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  		Figure 2. NPY reduces GABAA receptor-mediated inhibitory postsynaptic currents in parvocellular PVH neurons by a presynaptic action. A, The GABAA receptor blocker, picrotoxin (50 µM), inhibits most of the synaptic current evoked by electrical stimulation in a parvocellular PVH neuron. B, In the same neuron as in A, application of the AMPA-receptor antagonist, NBQX (1 µM), only slightly alters the evoked synaptic current. C, The application of NPY (300 nM) reversibly reduces the amplitude of the GABAergic IPSC in a parvocellular neuron. Synaptic responses in control, and in the presence of 300 nM NPY, are shown superimposed. D, NPY does not affect the change in membrane conductance caused by application of the GABAA agonist, muscimol (5 µM), to the cell in C. Shown superimposed are the membrane current responses to slow voltage ramps with a simultaneously applied train of brief 10 mV voltage steps, acquired in the presence of muscimol, in the absence and presence of 300 nM NPY. Vertical arrow indicates membrane current at the potential used to study IPSCs in this neuron.

 
As we previously reported (6, 24), bath application of NPY caused a reduction in the amplitude of evoked IPSCs (Fig. 2CGo), which reversed upon peptide washout. NPY inhibited the IPSC in almost all neurons tested. The peak of the effect was observed within 3–4 min of NPY application. At 500 nM, NPY reduced the IPSC amplitude by 57.1 ± 3.6 (n = 7, P < 0.0001, paired t test). The inhibition reversed within 25–40 min of washout. The action of NPY appeared to be entirely presynaptic, as no changes were observed either in the holding current or in the membrane current response to the voltage steps (Fig. 1CGo, inset). NPY also did not significantly alter the postsynaptic response to application of the GABAA agonist, muscimol (5 µM; n = 6, Fig. 2DGo).

To determine the apparent affinity of NPY at the presynaptic receptor, we tested NPY in the concentration range between 10 nM and 1 µM. NPY was less effective at 1 µM than at 500 nM, giving the concentration-response curve an inverted "U" shape, and therefore the data from this high concentration were excluded from the analysis. The EC50 determined from the dose-response data were about 28 nM (Fig. 3Go).



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  		Figure 3. Concentration-response curve for the NPY-mediated inhibition of the IPSC in parvocellular PVH neurons. The EC50, calculated from all concentrations of NPY below 1 µM, was 28 nM. Data are all from cells in which the effect of NPY reversed substantially on washout.

 
Structure-activity relationship of the NPY response in mpPVN
Because the pharmacology of the feeding response to NPY had been characterized on the basis of agonist actions, we next compared the sensitivity of the IPSC to other NPY agonists, including many that have been tested in feeding experiments in vivo (14, 17). We tested an array of NPY agonist analogues, whose affinities have been characterized at several cloned NPY receptor subtypes (Table 1Go). In these experiments, all agonists were tested at several different concentrations, but we compared the effects of them all at a concentration of 500 nM because of the small effects of some agonists tested at lower concentrations (Fig. 4Go).


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  		Table 1. Affinity constants (Ki) for the agonists (upper section) and antagonists (lower section) used in this study

 


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  		Figure 4. Structure-activity relationship of NPY and related agonists on the inhibition of the IPSC in rat parvocellular PVH neurons. All compounds were tested at 500 nM. At this concentration, all agonists had significant effects on the IPSC except rat PP. Numbers at the base of each barindicate the number of neurons responding to the application of different concentrations of the respective agonist/number of neurons tested. Numbers at the top of each bar indicate the number of cells tested at 500 nM. Symbols above bars indicate significance of agonist response against control: *, P < 0.001, {diamondsuit}, P < 0.01, {bullet}, P > 0.05.

 
PYY, a naturally occurring peptide, acts at all known NPY receptors, except Y3 (13, 35). Bath application of PYY inhibited the IPSC significantly, but with less effect than NPY. NPY2–36, a particularly effective agonist of the feeding response to NPY (14), also inhibited IPSCs in nearly all cells tested at different concentrations, but was less effective than NPY at 500 nM. The Y1- and Y5-preferring agonist, Leu31, Pro34NPY (Table 1Go), was also effective in nearly all experiments, as was the centrally truncated, Y1 and Y5 receptor agonist, [ahx8–20]Pro34NPY (36).

The Y2-selective, centrally truncated agonist, [ahx5–24]NPY, which has no activity at Y1 or Y5 receptors (32, 37), reduced the IPSC amplitude in 8 of 12 neurons tested. The Y5- and Y2- preferring agonist, D-Trp32NPY20, inhibited IPSCs significantly, but in only 50% of the neurons tested. The recently developed, highly selective Y5 agonist, Ala31Aib32NPY31, which has no significant affinity for the Y1, Y2 or Y4 receptors (Table 1Go), also reduced the IPSC in half of the cells tested. hPP was less effective than the other agonists but was active in most cells tested. By contrast, the Y4-specific agonist, rat PP, did not significantly inhibit the IPSC, and only 4 of 9 cells responded measurably. In all, none of the agonists tested at 500 nM were as effective as NPY.

Thus, all GABAergic inputs to mpPVN neurons responded to NPY or to any NPY-related agonist with an inhibition of IPSC amplitude. However, depending on the agonist, the percentage of cells responding and the magnitude of the response varied considerably. Based on these results, we concluded that Y1, Y2, and Y5 receptors, or a combination of these, could underlie the in vitro response to NPY itself, whereas neither Y4 nor Y3 receptors are likely to participate significantly. However, as there was no clear indication that any one of the functional receptor subtypes contributed more than the others, we next used receptor-selective antagonists to dissect the pharmacology of the response to NPY.

Y1, Y2, and Y5 receptors contribute to the action of NPY in mpPVN
In all of the remaining experiments, we first determined that the IPSC of a neuron responded to the test agonist, before trying the antagonist. To test the contribution of Y1 receptors, we compared the effect of NPY on the IPSC of individual mpPVN neurons in the absence and presence of a Y1 receptor antagonist. In this series of experiments, 100 nM NPY reduced the IPSC by 57.0 ± 3.8%. This effect was significantly reduced to about 65% of the control NPY effect when repeated in the presence of the Y1 antagonist BIBP 3226 (Fig. 5AGo). However, BIBP3226 counteracted the effect of NPY in only 7 of 13 neurons tested; in the remainder, there was no significant effect of BIBP3226 on the actions of NPY.



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  		Figure 5. The effect of Y1-, Y5-, and Y2-selective antagonists on the NPY- and specific agonist-mediated presynaptic inhibition of the IPSC in parvocellular neurons. A, Bars represent the effect of NPY in the presence of selective antagonists, normalized to the control effect of NPY alone in the individual cells tested. B shows the relative effect of specific agonists in the presence of selective antagonists, normalized to the effect of the given agonist alone in the individual cells tested. All data are from neurons in which NPY or a given agonist was tested first in the absence and later in the presence of an antagonist, and which demonstrated substantial recovery from the agonist under all conditions applicable. Symbols above bars indicate significance of agonist response in the presence of antagonist against control: {diamondsuit}, P < 0.01; {bullet}, P > 0.05.

 
To investigate the contribution of Y5 receptors, we studied the action of NPY in the presence of a Y5 receptor antagonist, Novartis2 (38). NPY (300 nM) alone decreased the amplitude of the IPSC by 43.5 ± 2.9%, and, on average, this effect was reduced in half in the presence of 500 nM Novartis2, in all cells tested (Fig. 5AGo). In light of the relatively incomplete antagonism of the action of NPY by either the Y1 or Y5 antagonists, we next tested the hypothesis that Y2 receptors may contribute to the effect of NPY. We therefore compared the effects of NPY (100–300 nM) in the absence and presence of the Y2-specific blocker, BIIE0246 (100–300 nM). In 4 of 11 cells tested, BIIE0246 completely prevented the effect of NPY, whereas in the remaining 7 neurons the antagonist substantially reduced the effect of NPY. On average, BIIE0246 reduced the effect of NPY on the IPSC by 80% (Fig. 5AGo). Thus, while there was evidence for all three receptor subtypes contributing to the effect of NPY in mpPVN, no single subtype contributed exclusively to the majority of NPY responses there.

Therefore, we next hypothesized that more than one NPY receptor subtype may contribute to the inhibition of GABA release from terminals on mpPVN neurons. We compared the action of NPY (300 nM) in the absence or presence of a combination of the Y1 and Y5 blockers, BIBP3226 (500 nM) and Novartis2 (500 nM). In this case, we observed a large reduction in the inhibitory action of NPY, by about 90%, consistent with a role for multiple NPY receptors (Fig. 5AGo).

Do "receptor-preferring" agonists act at their "preferred" receptors in mpPVN?
Because much of the in vivo data on the NPY receptor subtype mediating feeding is based on responses to agonist analogs and fragments, we established the receptor specificity of the subtype-preferring agonists in our preparation, using receptor-specific antagonists.

We first tested the effects of the Y1 antagonist on the actions of the prototypical Y1 agonist, Leu31, Pro34NPY. In this series of neurons, Leu31, Pro34NPY (500 nM) inhibited the IPSC by 35.8 ± 6.5%, but in the same neurons, this effect was reduced on average by over 30% in the presence of 500 nM BIBP3226 in all cells tested (Fig. 5BGo). Against [ahx8–20]Pro34NPY (500 nM), BIBP3226 (500 nM) reduced the effect on the IPSC on average by over 70% in 9 of 13 neurons tested, blocking it entirely in 4 of these cells (Fig. 5BGo). BIBP3226 (500 nM) had no effect on the responses of 4 cells to [ahx8–20]Pro34 NPY. BIBP3226 also did not affect the action of the Y5/Y2 agonist D-Trp32 NPY in 4 cells tested (Fig. 5BGo).

The reduction of the NPY effect on the IPSC by the Y5-selective antagonist, Novartis2, suggested the presence of Y5 receptors on some inputs to these neurons. We therefore tested the actions of the Y5-selective antagonists against Y5-preferring agonists. First, we tested whether the actions of D-Trp32NPY were sensitive to a Y5 antagonist. Novartis2, applied at 500 nM, reduced the effect of D-Trp32NPY (500 nM) by about 90% in all neurons tested (Fig. 5BGo). A different Y5 antagonist, Novartis1 (500 nM) reduced the effect of the more potent and highly selective Y5 agonist, Ala31Aib32NPY by about 85% on average in all 5 neurons tested (Fig. 5BGo). The effect of Novartis2 (500 nM) was tested on 11 neurons where the IPSC was sensitive to the mixed Y1/Y5 agonist Leu31Pro34NPY (500 nM). The antagonist was effective in 8 neurons, blocking the response completely in 4 of these cells, and reducing it on average by 70% overall. The responses of 3 cells were unaffected by Novartis2. We also examined the effects of the Y2 antagonist, BIIE0246 on the IPSC inhibition mediated by the Y2-selective agonist, [ahx5–24]NPY. The antagonist reduced the effect of this agonist by over 90% in all 4 neurons tested.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
While the role of NPY in natural feeding remains controversial, it is an extremely potent orexigen (1). Its expression in the hypothalamus is regulated by energy state (39), it is coexpressed by ARC neurons that are inhibited by the anorectic signalers, {alpha}-melanocyte-stimulating hormone (MSH) and leptin (40), all of which is consistent with a central role in energy balance. Our aim in these experiments was to identify the receptors mediating the response to NPY in mpPVN that may underlie at least part of its powerful effects on food intake. In several hundred conventional whole-cell patch clamp recordings from mpPVN neurons, the only response we observed to application of NPY and related agonists was a prominent suppression of a GABAA-mediated IPSC. This effect was seen in nearly every neuron tested with NPY itself. The pharmacology of this NPY response in vitro is broadly consistent, though not identical, with that of the in vivo feeding response and is consistent with the participation of multiple NPY receptor subtypes.

Our data supports an entirely presynaptic site of NPY action in the rat mpPVN because NPY affected neither the resting membrane current, the response to voltage steps or subthreshold voltage ramps, nor the membrane current response to the GABAA agonist, muscimol. Unlike in ARC neurons (41), here we found no evidence for NPY effects on postsynaptic membrane properties.

The structure-activity studies using receptor-preferring agonists were not consistent with the activation of a single known receptor subtype. NPY itself was the most potent and frequently active of the agonists, followed by PYY and the "feeding receptor" agonist, NPY2–36; these three are also the least receptor-selective of the agonists tested. The effectiveness of PYY is consistent with feeding data and makes the participation of Y3 receptors in the PVN response unlikely, whereas the ineffectiveness of rPP, even at a high concentration, makes it very unlikely that the Y4/PP1 receptor is involved. Rats, like humans, do not express the y6 receptor (42) so the only known receptors, which might be involved, are Y1, Y5, and Y2. However, despite the involvement of different receptors, NPY appears to have only a single kind of effect in mpPVN, the suppression of synaptic GABA release, at least under the conditions in this study.

Y1 receptors
Three lines of evidence support a role for Y1 receptors in the response of the IPSC to NPY. Agonists such as Leu31Pro34NPY and [ahx8–20]Pro34NPY prefer Y1 receptors about 10-fold over Y5, and about 10,000-fold over Y2 (Table 1Go). More importantly, blocking concentrations of the Y1-selective antagonist, BIBP3226, reduced the effects of NPY itself, albeit in only half of the cells tested. Finally, blocking concentrations of the Y5 antagonist, Novartis2, failed to affect the actions of the mixed Y1/Y5 agonist, Leu31Pro34NPY, in about one-third of neurons tested, and only reduced it in a further one-third of cells. Because Leu31Pro34NPY has no significant affinity for the Y2 receptor, these results are consistent with the Y1 receptor mediating a part of the effect of NPY on the IPSC. Interestingly, pNPY has a 10-fold higher affinity for rat Y1 receptors than does human (and rat) NPY (28), and pNPY is more potent at eliciting food intake in rats than is human NPY (14).

Although Leu31Pro34NPY and Pro34NPY, both Y1-preferring agonists, cause rats to eat when injected intracerebroventricularly (icv) (17), the evidence for Y1 receptors being involved in feeding is controversial. Some studies using the Y1 receptor-selective antagonists BIBP3226, BIBO3304 or 1229U91 reported inhibition of NPY-induced and spontaneous feeding (15, 16, 22, 43, 44), whereas other studies showed no effect of Y1 receptor antagonists (20, 42, 45). In the present study, Y1 receptors appeared to have a smaller role than Y5 or Y2 receptors in mediating the effects of NPY in the mpPVN.

Y5 receptors
Several lines of evidence also implicate Y5 receptors in the actions of NPY in mpPVN. Thus, the Y5- (and Y2-) preferring agonist, D-Trp32NPY and the highly Y5- selective agonist, Ala31Aib32NPY, were both as effective at inhibiting the IPSC, as was Leu31Pro34NPY. The Y5-selective antagonist, Novartis2, blocked the effects of both these Y5 agonists. Novartis2 also blocked the effects of Leu31 Pro34NPY in one-third of cells tested, and reduced its effect in a further one-third. Finally, Novartis2 always reduced the effects of NPY, although it never entirely prevented them. Interestingly, when the Y1 and Y5 antagonists were applied together, the effects of NPY were blocked.

There is considerable evidence for a role of Y5 receptors in feeding. Y5-preferring or -selective agonists stimulate feeding in rodents (17, 31, 43). Administration of Y5 antisense oligodeoxynucleotides decreased spontaneous food intake and inhibited the robust feeding response to NPY (46, 47). Finally, Y5-selective antagonists have been reported to antagonize NPY-induced feeding in rats (44, 48). Here although Y5 receptors appeared to play a major role in the inhibition of the IPSC, they were not the only receptors involved.

Y2 receptors
We found considerable evidence for a role of Y2 receptors in mediating the inhibition of the IPSC in mpPVN. The Y2- specific agonist, [ahx5–24]NPY, was comparably effective with the Y5-selective agonists in suppressing the IPSC, and was active at more neurons (at 75% vs. 50% for the Y5 agonists). The Y2 specific antagonist, BIIE0246, reduced the effect of NPY at all synapses tested, blocking it entirely at some. Indeed, the effect of BIIE0246 on the IPSC inhibition mediated by NPY was greater than that of the Y5 blocker, and much greater than that of the Y1 blocker BIBP3226 (which only had an effect in half the neurons tested). As expected, BIIE0246 entirely blocked the effect of the Y2-selective agonist, [ahx5–24]NPY.

There is relatively little evidence in support of a role of Y2 receptors in feeding. Thus, icv injections of Y2-selective agonists were ineffective in stimulating food intake (38, 42), including C2NPY, a related, centrally truncated Y2 agonist (17). Furthermore, icv injections of NPY were equally effective in wild-type and Y2-knockout mice (49). Y2-knockout mice developed mild obesity due to hyperphagia and reduced activity. However, Y2 receptors were localized to NPY-containing somata (50) and to NPY-containing terminals in the hypothalamic arcuate and PVN nuclei (49), suggesting they may modulate NPY release. Because GABA is also colocalized in a subset of arcuate NPY neurons (10), it is possible that some of the GABA responses we have observed in vitro may originate from these fibers, if they project to the PVN. Consistent with this, Y2 receptors have been shown to reduce NPY release in hypothalamic slices (51).

Does the electrophysiological response to NPY in PVN relate to feeding?
The PVN appears to be an important link in the pathway responsible for the regulation of natural eating behavior (1), and NPY appears to be important in this pathway for several reasons. 1) The PVN receives a rich NPY projection from the ARC (5, 8), concentrated in the parvocellullar region (52). 2) Alterations in NPY levels related to food deprivation and refeeding are restricted to the parvocellular PVN (53, 54). 3) Leptin, which suppresses feeding, also inhibits arcuate NPY neurons (40, 41, 55). 4) Both NPY ( 56, 57) and leptin (58) activate c-fos in the parvocellular division of PVN.

Any attempt to correlate in vivo feeding responses to responses in vitro in brain slices must be indirect. We believe that the NPY-mediated suppression of the IPSC in the mpPVN is related to feeding because: 1) this IPSC is enhanced by {alpha}-MSH, which is known to suppress feeding; 2) AGRP, a peptide contained in NPY projections from ARC to the PVN, and a potent orexigen, prevents the actions of {alpha}-MSH at these synapses (6); and 3) as we demonstrate here, the pharmacology of the NPY response is broadly consistent with the feeding data. It must be recognized here that neurons in a brain slice lack many of the connections they normally receive, and while many axons remain alive in the preparation, it cannot be excluded that some physiologically important inputs, even neurons, are systematically lost in the preparation, so the present experiments cannot exclude additional actions of NPY possibly lost in the preparation, which may be important in feeding. Nevertheless, despite the indirect nature of the evidence here, the correspondence between in vivo eating responses and in vitro physiological and pharmacological responses is strong. Finally, the fact that multiple NPY receptors participate in the regulation of the IPSC may explain why the knockout of any single NPY receptor has little effect on the feeding response to NPY itself.

There is some evidence that injection of muscimol into the lateral ventricles or directly into the PVN increases food intake in rats, and may be additive with the effects of some concentrations of NPY (59). GABA receptors are ubiquitous in the CNS, including the PVN, so it is unclear from this report which population of neurons are involved in these experiments, but it is unlikely to be the population of neurons reported earlier to be excited by NPY injections (56). It is clear that further work is needed to clarify the population of cells in mpPVN responsible for the orexigenic actions of NPY.

Other effects of NPY injection into the PVN include reductions in blood pressure and heart rate (60), the release of CRF resulting in ACTH release (61), the suppression of TRH release and TRH mRNA expression (62), and an increase in ethanol self-administration (63). Any or all of these actions might also be mediated by the same presynaptic mechanisms we describe here. A more definitive explanation awaits the identification of the neurochemical phenotype or phenotypes of the parvocellular neurons from which we recorded here.

Our results suggest that several NPY receptors mediate very similar responses on a potentially heterogeneous population of GABA terminals in the mpPVN, with a response profile broadly similar to that for stimulating appetite. It is tempting to speculate from this that the natural ligand, NPY, or exogenous, NPY-related ligands that activate multiple NPY receptors with high affinity, are the most active at eliciting the feeding response precisely because they act at more than one NPY receptor. The presence of Y2 receptor-mediated responses, while unlikely to be directly related to induction of feeding, is consistent with an autoreceptor-mediated inhibition of GABA release from NPY/GABA/AGRP fibers originating from the ARC. Two essential questions that remain are: what are the source(s) of the NPY-sensitive GABA inputs to mpPVN, and what are the neurochemical phenotypes of the parvocellular neurons whose inputs are sensitive to NPY.


   Acknowledgments
 
We are grateful to Drs. P. Hipskind, Lilly Research Laboratories, for his gift of Y5 antagonists, to Dr. Henri Doods, Boehringer Ingelheim GmbH, for his gift of BIIE0246, and to Drs. Roger Cone and Michael Cowley for their comments on an earlier version of the manuscript.


   Footnotes
 
This work was Supported by the Medical Research Council (Canada)-Pharmaceutical Manufacturers Association of Canada program (Eli Lilly Canada, sponsor).

Abbreviations: aCSF, Artificial cerebrospinal fluid; AGRP, agouti gene-related peptide; AMPA, {alpha}-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; ARC, arcuate nucleus; hPP, human PP; icv, intracerebroventricular(ly); IPSC, inhibitory postsynaptic current; mpPVN, medial parvocellular region of the PVN; MSH, {alpha}-melanocyte-stimulating hormone; NBQX, 6-nitro-7-sulfamoylbenzo (f)quinoxaline-2,3-dione; PP, pancreatic polypeptide; pNPY, porcine NPY; PVN, paraventricular nucleus; PYY, peptide YY.

Received July 27, 2001.

Accepted for publication October 23, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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