Neuropeptide Y and epilepsy: varying effects according to seizure type and receptor activation

Neuropeptide Y and epilepsy: varying effects according to seizure typeand receptor activation

Sophie Reibel*,a Suzan Nadib, Ramla Benmaamara, Yves Larmetc, Josette Carnahand,Christian Marescauxa, Antoine Depaulisa

aINSERM U398, Faculte´ de Medecine, 11 rue Humann, 67085 Strasbourg cedex, FrancebNaval Medical Research Institute, National Naval Medical Center, Bethesda, MD 20889-5607, USA

cCNRS-UMR 7519, Universite´ Louis Pasteur, 21 rue Rene´ Descartes, 67084 Strasbourg cedex, FrancedAmgen Center, 1840 DeHavilland Drive, Thousand Oaks, Los Angeles, CA 91320-1789, USA

Received 7 August 2000; accepted 15 November 2000

Abstract

In vitro andin vivo experiments suggest antiepileptic properties for NPY. In this study, the pharmacology of these effects was examinedand compared in different rat models of seizures. Agonists for Y1, Y2 and Y5 receptors reduced seizure-like activity in hippocampal cultures.Intracerebral injection of NPY or Y5 agonists reduced the expression of focal seizures produced by a single electrical stimulation of thehippocampus. Conversely, NPY agonists increased the duration of generalized convulsive seizures induced by pentylenetetrazol. Theseresults suggest that NPY reduces seizures of hippocampal origin through activation of Y5 receptors. They also point to probable modulatoryeffects of NPY in brain structures other than the hippocampus, involved in initiation, propagation or control of seizures. © 2001 Publishedby Elsevier Science Inc.

Keywords:Electrical stimulation; Epilepsy; Hippocampus; Hippocampal cell culture; Neuropeptide Y; Neuropeptide Y receptors; Pentylenetetrazol; Rat;Seizures; Y1; Y2; Y5

1. Introduction

A growing body of data suggests the involvement ofneuropeptide Y (NPY) in the modulation of epileptic sei-zures [74]. Moreover, the hippocampus appears as a targetstructure for the modulatory effects of this peptide on sei-zures. In this structure, the expression of NPY is stronglyincreased in different models of epilepsy [42,49,63,64,66],where seizures induce an overexpression of NPY inGABAergic interneurons [12,64] andde novosynthesis indentate granule cells and release by mossy fibers [44,59].Furthermore, the seizure-related increase in NPY expres-sion is accompanied by modified levels of NPY receptor

subtypes Y1, Y2 and Y5 in the hippocampus [7,28,36,37,57,62,72].

Recent evidence fromin vitro and in vivo experimentssuggests that NPY has suppressive effects on epileptic sei-zures. In human and rodent hippocampal slices, NPY re-duces neuronal excitability [1,4,9,10,34,35,46,53,65].Transgenic mice which do not express NPY are more sus-ceptible to generalized convulsive seizures induced by sys-temic injection of pentylenetetrazol or kainic acid [1,13,20],and intracerebroventricular injection of NPY reduces sei-zure activity in the rat [76,77]. However, thesein vivostudies mainly used models of generalized convulsive sei-zures, which do not allow to address the question of thehippocampus as a possible target for NPY’s effects. To date,the pharmacological profile of these effects has principallybeen investigatedin vitro. In the hippocampal slice, NPYappears to reduce neuronal excitability through presynapticactivation of Y2 receptors [9,30,35,46,54].In vivo, only onesystematic pharmacological study has been performed anddescribes anticonvulsant properties of NPY secondary to Y5

receptor activation on kainic acid-induced seizures [76]. Y5

Abbreviations: EEG5 electroencephalogram; ICV5 intracerebrov-enticular; IH5 intrahippocampal; IP5 intraperitoneal; NPY5 neuropep-tide Y; PBS5 phosphate buffered saline; TPP1 5 tetraphenylphospho-nium.

* Corresponding author. Tel.:133-4-78-78-57-06; fax:133-4-78-77-86-16.

E-mail address:[email protected] (S. Reibel).

Peptides 22 (2001) 529–539

0196-9781/01/$ – see front matter © 2001 Published by Elsevier Science Inc.PII: S0196-9781(01)00347-3

receptor knock-out mice are also more sensitive to kainateseizures [43] and activation of Y2/Y5 receptors reduces theenhanced susceptibility to pentylenetetrazol secondary tokainic acid status epilepticus [73]. Finally, proconvulsantproperties of Y1 receptor activation have been suggestedfrom the inhibitory effects of a Y1 antagonist in the kainicacid model [23].

In this study, the pharmacological profile of the effects ofNPY on seizures was further characterizedin vitro and invivo. The most commonly used agonists for the Y1, Y2 andY5 receptors were used in the absence of selective and highaffinity agonists.In vitro, epileptiform activity was inducedin primary hippocampal cell cultures by exposure then with-drawal of a glutamate antagonist [21]. In order to examinewhether modulatory properties of NPY on seizures are spe-cific to the hippocampus, two models of epilepsy differen-tially involving the hippocampal formation were usedinvivo: focal hippocampal seizures induced by electrical stim-ulation of the hippocampus and generalized convulsive sei-zures induced by systemic injection of pentylenetetrazol. Inthese models, the effects of intracerebroventricular and bi-lateral intrahippocampal injections were compared.

2. Materials and methods

2.1. In vitro experiments

2.1.1. Hippocampal cell culturesPrimary cultures of hippocampal cells were carried out

as previously described [61]. Hippocampus from E20–E21rat pups was dissected and dissociated using a papain-deoxyribonuclease mix. Individual cells were grown at adensity of 400,000 in 35 mm Petri dishes coated withpolyornithine (15mg/ml), in defined neurobasal mediumsupplemented with B27 (GIBCO BRL) and 0.5 mM glu-tamine (Sigma) (37°C, 5% CO2). After two weeks in cul-ture, the cells were stained with anti-synaptophysin [51] andthe neuronal specific antibody anti-TuJ1 recognizing classIII beta-tubulin [25].

2.1.2. Treatment of cultures with peptides and inductionof epileptiform activity

Hippocampal cultures were treated for 48 hours withporcine NPY (0.24 nmol/ml), human [Leu31Pro34]PYY(0.24 nmol/ml), human PYY3-36 (0.12 nmol/ml) or humanPP (0.24 nmol/ml), in culture medium with leupeptin (0.5mg/ml, Sigma) in order to prevent degradation. Controlcultures were treated with medium and leupeptin only. Par-oxysmal activity was induced by exposure (24 hours, on thesecond day of peptide treatment) and then withdrawal of aionotropic glutamate antagonist (MK801, 10mM, Sigma)[21]. Removal of the excitatory synaptic blocker leads toseizure-like activity consisting of paroxysmal depolariza-tion shifts. Quantitative evaluation of the seizure-like activ-ity was performed by measuring the membrane potential

shift induced by the paroxysmal neuronal activity. Thisevaluation was based on a simple and validated methoddeveloped by Lichtshtein et al. (1979), in which the mem-brane potential variation is estimated from the extracellularto intracellular ratio of [3H]-tetraphenylphosphonium(TPP1). TPP1 is a non toxic lipophilic cation that crossesthe neuronal membrane and distributes itself according tothe Nernst equation. A low intracellular level of TPP1

indicates a depolarization and increased level a hyperpolar-ization. The exact membrane potential can be calculated byinserting the intracellular and extracellular concentrations ofTPP1 into the Nernst equation. It has been shown previ-ously that the results obtained from TPP1 measurementsagree closely with the intracellular electrophysiologicalmeasurements carried out in sister cultures [41]. In thisstudy, cell cultures were equilibrated with 2mM [3H]-TPP1

(10,000 dpm/mmol) (Amersham) for 20 minutes at 37°Cbefore induction of epileptiform activity. Thirty minutesafter the beginning of paroxysmal activity, supernatant (ex-tracellular) and cell lysat (intracellular) [3H]-TPP1 concen-trations were measured by liquid scintillation counting. Us-ing the Nernst equation, membrane potential variationswere calculated in 6 different cultures for each agonisttested and expressed as absolute values (mV).

2.2. In vivo experiments

2.2.1. AnimalsAdult male Wistar rats weighing 300 to 350 g were used.

Once weaned, they were housed in groups of 8–12 malesuntil surgery. After surgery, they were placed in individualPlexiglas cages and received food and waterad libitum. Allanimal experiments were carried out in accordance with theEuropean Community Council Directive of 24 November1986 (86/609/EEC) and with the principles set forth in theGuide for the Care and Use of Laboratory Animals, Instituteof Laboratory Animal Resources, National Research Coun-cil, National Academy Press, 1996.

2.2.2. SurgeryAnimals were premedicated with diazepam (4 mg/kg,

intraperitoneal (IP)) and anaesthetized with ketamine (100mg/kg, IP). They were placed in a stereotaxic frame in flatskull position. For intracerebral injections, stainless steelguide cannulae (0.3 mm inside diameter, 0.4 mm outsidediameter) were implanted in the right lateral ventricle (an-teroposterior 0.8 mm, mediolateral 1.3 mm, dorsoventral3.7 mm, with bregma as reference), or in the dentate gyrusof the dorsal hippocampus bilaterally (anteroposterior 4.0mm, mediolateral 2.0 mm, dorsoventral 3.7 mm, withlambda as reference). Stainless steel stylets were inserted inthe cannulae to prevent their obstruction. Three monopolarcortical electrodes were screwed into the skull above the leftfrontoparietal cortex and the right frontal cortex. For elec-trical stimulation of the hippocampus, a bipolar electrodemade of two twisted enamel-insulated wires (0.18 mm in

530 S. Reibel et al. / Peptides 22 (2001) 529–539

diameter) was implanted in the dentate gyrus of the rightdorsal hippocampus (same coordinates as above). For ani-mals receiving bilateral intrahippocampal injections, thebipolar electrode was glued to the right-hand side cannulawith cyanoacrylate so that the distal tip of the electrode waslocated 1 mm below the tip of the cannula. All electrodeswere connected to a female connector placed over the skulland secured with acrylic cement. Animals were allowed aweek for recovery during which time they were handleddaily for habituation.

2.3. Seizure models

2.3.1. Focal seizures induced by electrical stimulation ofthe hippocampus

The animals were stimulated through the bipolar elec-trode using a monophasic square wave current (frequency550 Hz, duration5 2 s, pulse5 1 ms). The afterdischargethreshold was determined by increasing the current intensityby 25 mA steps every two minutes until an afterdischargecould be recorded on the electroencephalogram (EEG). Cor-tical and hippocampal primary afterdischarge durationswere measured on the EEG trace. EEG recording was car-ried on for a further three minutes following the end of theprimary afterdischarge. When a secondary afterdischargeoccurred during this time, its duration was measured on thecortical EEG and the number of whole body shakes or “wetdog shakes” accompanying it were counted. Stimulationsessions were separated by at least a week and limited to amaximum of four to avoid a kindling effect.

2.3.2. Generalized convulsive seizures induced bypentylenetetrazol

Generalized clonic seizures were induced by injection ofpentylenetetrazol (45 mg/kg, IP) dissolved in sterile dis-tilled water. Latency to first seizure occurrence and cumu-lative seizure duration (i.e., sum of the durations of indi-vidual seizures) during a one-hour-period following theinjection were measured on the cortical EEG. Injections ofpentylenetetrazol were spaced out by at least a week andeach animal received a maximum of four injections to avoida kindling effect.

2.4. Intracerebral injections

Acute intracerebral injections were performed 30 min-utes prior to seizure induction by pentylenetetrazol or 20minutes before electrical stimulation of the hippocampus.For intracerebroventricular (ICV) injection of NPY or NPYreceptor agonists, an injection cannula (0.18 mm insidediameter, 0.28 mm outside diameter) connected to a 10mlmicrosyringe moved by a microinjection pump (CarnegieMedicin) was inserted in the guide cannula. The rats re-ceived 12 nmol of human NPY, human [Leu31Pro34]PYY(preferential Y1 agonist with affinity for Y5), humanPYY3-36 (preferential Y2 agonist with affinity for Y5),

porcine NPY13-36 (preferential Y2 agonist), human PP (Y5agonist) (Amgen, Thousand Oaks, USA) or vehicle (NaCl0.9% with 1% bovine serum albumin), in a volume of 5mlover two minutes [6,26,48]. These doses have previouslybeen shown to modulate seizure expression [76,77]. Forbilateral intrahippocampal injections, vehicle or 0.8 nmol/side of NPY or NPY agonists were administered in a vol-ume of 0.4ml/side over two minutes. Lower doses of NPYor [Leu31Pro34]NPY have been shown to have little or noeffect on seizures [23,65, personal observation]. For bothICV and intrahippocampal injections, the injection cannulawas removed one minute after the end of the injection andreplaced by the stylet. Animals were injected with vehicle orone of the agonists in a counterbalanced order with oneweek between two injections. Food pellets were placed inthe EEG recording cage and presence or absence of feedingduring the hour following the intracerebral injection wasnoted by the experimenter.

2.5. Histological controls

At the end of the experiments, animals were sacrificedwith an overdose of pentobarbital (200 mg/kg, IP). Brainswere frozen and 20mm cryostat sections were stained withCresyl Violet. The position of the cannulae and stimulatingelectrodes was determined histologically and only rats withcorrect locations in the lateral ventricle or the dorsal hip-pocampus were included for statistical analysis.

2.6. Statistical analysis

Comparison ofin vitro effects was performed using theStudent t-test for paired values. Seizure parameters col-lected from thein vivo models were compared using ananalysis of variance withpost hocBonferroni’s test. For allexperiments, statistical significance was set atp , 0.05.

3. Results

3.1. Effects of NPY agonists on seizure-like eventsproduced in hippocampal cell cultures

The effects of different NPY agonists on seizure-likeactivity were tested at the cellular level in primary hip-pocampal cell cultures. After two weeks in culture, visual-ization of hippocampal cells with anti-TuJ1 and anti-synap-tophysin antibodies confirmed extensive neuronal networksand synapse formation, respectively. It has been describedpreviously that following exposure then withdrawal ofMK801, a ionotropic glutamate antagonist, the cells displaya paroxysmal depolarization shift, with repetitive actionpotential firing [21]. A 48-hour application of NPY (0.24nmol/ml), [Leu31Pro34]PYY (0.24 nmol/ml), PYY3-36(0.12 nmol/ml), or PP (0.24 nmol/ml) significantly reduced

531S. Reibel et al. / Peptides 22 (2001) 529–539

the membrane potential shift following MK801 withdrawal(Fig. 1).

3.2. Effects of intracerebral injection of NPY agonists onthe general behavior of the rats

ICV injections of the different NPY agonists did notaffect locomotor activity or arousal status of the animals,nor did it modify the EEG pattern recorded for 30 minutesafter injection of the peptides and before seizure induction.However, ICV injections of NPY, PYY3-36 and PP (12nmol) invariably induced a feeding behavior with compul-sive chewing of food pellets, also evidenced on the corticalEEG recording by masticatory artefacts. Animals startedeating 10 to 15 minutes after the injection. Feeding wasinterrupted by seizure activity but usually recurred after-wards, lasting for 60 to 75 minutes in total. ICV injectionsof [Leu31Pro34]PYY or NPY13-36 did not systematicallyinduce such a feeding behavior, and animals injected withvehicle never ate during the observation period.

Following intrahippocampal peptide injection, no wetdog shakes or other behavioral or EEG modifications wereobserved. Rats treated with NPY, [Leu31Pro34]PYY,PYY3-36 and PP were only occasionally seen eating, andNPY13-36 never induced a feeding behavior.

3.3. Effects of NPY agonists on focal hippocampalseizures induced by electrical stimulation of thehippocampus

The effects of intracerebral application of NPY agonistswere studied in a model of focal hippocampal seizures. Lowintensity electrical stimulation of the hippocampus (25–100mA) induces a local epileptic activity, with limited propa-gation to other brain areas [29]. This epileptic activity isobserved on the EEG recording as an afterdischarge char-acterized by a high amplitude spiking activity. Behaviorally,the animals remain immobile. Within one or two minutesafter the end of this primary afterdischarge, a secondaryafterdischarge may occur, often accompanied by repetitivewhole body shakes described as “wet dog shakes”.

Following ICV injection of NPY, [Leu31Pro34]PYY,PYY3-36, NPY13-36 or PP (12 nmol), afterdischargethresholds were not modified (results not shown). However,all compounds reduced the durations of primary and sec-ondary afterdischarges. This reduction reached significancefor secondary afterdischarges after injection of PYY3-36and PP. In addition, the number of wet dog shakes wassignificantly reduced by NPY and PP, and completely abol-ished by PYY3-36 (Fig. 2).

In order to examine whether these inhibitory propertiesresult from an action of NPY agonists within the hippocam-pus, the effects of intrahippocampal injections were studied.Bilateral injections were performed as both hippocampiappear to be involved in the generation of an afterdischarge[70]. Bilateral intrahippocampal injections of vehicle, NPY,[Leu31Pro34]PYY or PYY3-36 (0.8 nmol) did not modifyafterdischarge thresholds (results not shown) and primarycortical afterdischarge durations (Fig. 3). NPY completelyabolished secondary afterdischarges and wet dog shakes,whereas PYY3-36 significantly reduced only the number ofwet dog shakes. [Leu31Pro34]PYY increased primary hip-pocampal afterdischarge duration, without significantly af-fecting the other parameters (Fig. 3).

3.4. Effects of NPY agonists on pentylenetetrazol-inducedgeneralized convulsive seizures

The effects of NPY agonists, injected at the same dosesand via the same routes as in the hippocampal stimulationexperiment, were studied in a model of generalized convul-sive seizures induced by pentylenetetrazol, in which thehippocampus does not appear critical for seizure develop-ment [22]. Following injection of pentylenetetrazol (45 mg/kg, IP), all animals displayed from one to three generalizedconvulsive seizures, with strong clonic movements of the

Fig. 1. Effects of NPY agonists on membrane potential variation in primaryhippocampal cultures, measured by depolarization-dependent TPP1 incor-poration into cells (n 5 6 per drug tested). *p , 0.05.


fore and hind limbs. Occasionally, some rats also developeda tonic seizure with extension of the four limbs. None of theagonists injected modified the clinical presentation of theseizures (i.e., clonic versus tonic), but ICV injection of allcompounds (12 nmol) increased the cumulative seizure du-

ration, except for PP (Fig. 4). The number of seizures andlatency to first seizure were not significantly modified (re-sults not shown). Two animals treated with PYY3-36 de-veloped an electroencephalographic status epilepticuswithin two minutes following pentylenetetrazol injection,

Fig. 2. Effects of ICV injections of NPY agonists (12 nmol) on electroencephalographic and behavioral expression of seizures induced by electricalstimulation of the dorsal hippocampus in two series of experiments;n 5 9, for the group of animals treated with vehicle, NPY, [Leu31Pro34]PYY andPYY3-36; n 5 8, for the group of animals treated with vehicle, NPY13-36 and PP. WDS, wet dog shakes. *p , 0.05.


evidenced by continuous and regular spiking activity ac-companied by intermittent myoclonic jerks of the wholebody and lasting for the entire hour of recording. Theseanimals were not included in the analysis as their EEGactivity was different from the others.

Bilateral intrahippocampal injection of [Leu31Pro34]PYY

(0.8 nmol) significantly reduced latency to first seizure(81 6 4 s, versus 2836 152 s after vehicle injection,n 56), but did not modify cumulative seizure duration. NPYand PYY3-36 (0.8 nmol) injected in the hippocampus hadno effect on the expression of pentylenetetrazol-inducedconvulsive seizures (latency to first seizure, cumulative sei-

Fig. 3. Effects of bilateral intrahippocampal injections of NPY agonists on electroencephalographic and behavioral expression of seizures induced byelectrical stimulation of the dorsal hippocampus (n 5 7). WDS, wet dog shakes. *p , 0.05.


zure duration, clinical expression of the seizures) (resultsnot shown).

4. Discussion

4.1. NPY inhibits seizures of hippocampal origin

The in vitro and in vivo observations described in thepresent study suggest that NPY exerts antiepileptic effectsspecific to seizures initiated in the hippocampus (see Table1).

At the cellular level, NPY reduced the membrane poten-tial shift triggered by withdrawal from the culture mediumof a glutamate antagonist, and reflecting reduced seizure-like activity in the dissociated cell cultures [21]. This is thefirst description of inhibitory effects of NPY agonists onneuronal hyperexcitability in dissociated cell cultures. Sucha suppression of epileptiform activity by NPYin vitro hasalso been described in hippocampal slices, in which intrinsicneuronal networks are preserved [4,9,35]. This inhibitoryaction of NPY in two in vitro models characterized by

different degrees of hippocampal connectivity prompted usto examine the effects of the peptidein vivo, in experimentalmodels differentially involving the hippocampus for seizureinitiation.

When the hippocampus is electrically stimulated atthreshold intensity, focal seizures are produced with limitedpropagation to other brain sites [29], allowing to studyhippocampal excitabilityin vivo. In such a paradigm, asingle afterdischarge elicited in the hippocampus enhancesthe neuronal release of NPY in this structure, suggesting arole for this neuropeptide in the modulation of hippocampalexcitability [56]. In our study, ICV injection of NPY ago-nists decreased the duration of afterdischarges and the num-ber of wet dog shakes. To further examine the hypothesis ofan action of NPY on hippocampal excitability, the effects ofintrahippocampal injections of the peptide were studied.Injections were performed bilaterally, as activation of bothhippocampi participates in the induction of an afterdis-charge [16,70]. Intrahippocampal injection of NPY com-pletely suppressed secondary afterdischarges and wet dogshakes, but had no effect on primary afterdischarges. Thesecondary afterdischarge is thought to result from a rever-berating activity involving the entorhinal cortex and circu-lating back to the hippocampus [40]. Wet dog shakes, thebehavioral counterpart of this electroencephalographic ac-tivity [39], appear to be a motor activity initiated within thehippocampus [11,38,58]. Therefore, diminution or suppres-sion of these parameters can be considered to reflect de-creased propagation of seizure activity from the hippocam-pus. Observations similar to ours were reported followingICV injection of human NPY at the same dose [77]. In thisstudy, an increase of the afterdischarge threshold and areduction in the duration of the primary hippocampal after-discharge were also described. These discrepancies may berelated to (i) a different biodisponibility of the drugs usedwithin brain structures, (ii) differences in the stimulationparadigms used (in particular using 25mA rather than 50mA increments to determine seizure threshold) or (iii) dif-ferent genetic background of the animals which has beenshown to influence hippocampal excitability [45,70,77].Our results show that NPY reduces seizures of hippocampalorigin in vivo. They are in agreement with the observation

Fig. 4. Effects of ICV injections of NPY agonists (12 nmol) on thecumulated duration of generalized clonic seizures occurring during onehour after injection of pentylenetetrazol (45 mg/kg, IP). In brackets, num-ber of animals per experimental group. *p , 0.05.

Table 1Summary of the effects of NPY agonists on seizuresin vitro and in vivo

Agonist Preferentialbinding

Epileptiform activityin vitro

Hippocampalstimulation

Pentylenetetrazol

ICV IH ICV IH

NPY 2 2 2 1 Ø[Leu31Pro34]PYY Y1 2 Ø 1 1 1PYY3-36 Y2/Y5 2 2 2 1 ØNPY13-36 Y2 ND Ø ND Ø NDPP Y5 2 2 ND Ø ND

1 increased seizure activity;2 reduced seizure activity; Ø no effect; ND not done.ICV intracerebroventricular injection, IH intrahippocampal injection.


that NPY reduces the epileptic behavior induced by intra-hippocampal picrotoxin injection [65] and that ICV injec-tion of NPY diminishes hippocampal EEG activity inducedby systemic kainic acid administration and generalized sei-zures consequently [76].

4.2. NPY aggravates clonic seizures induced bypentylenetetrazol

Conversely, NPY injected in the hippocampus did notaffect generalized clonic seizures induced by systemic in-jection of pentylenetetrazol (45 mg/kg), a model in whichthe hippocampus does not appear to be critical for thegeneration and propagation of seizures [22,50]. Althoughexpression of NPY is increased in the hippocampus follow-ing generalized convulsive seizures induced by pentyle-netetrazol [44,67], our results suggest that this hippocampalmodulation of NPYergic activity does not affect that par-ticular type of seizure. On the contrary, NPY injected in theventricular system increased the duration of clonic seizuresinduced by pentylenetetrazol. This aggravating effect is inopposition with anticonvulsant properties reported for NPYon tonic seizures elicited by pentylenetetrazol (50–60 mg/kg) following ICV administration [75] and in NPY knock-out mice [20]. In another study, the severity of the seizureselicited by pentylenetetrazol was not modified in rats treatedin the hippocampus with anti-NPY antibodies [71]. Clonicand tonic seizures are underlain by different brain structures[22]. The differing effects of NPY in models of epilepsywith distinct anatomical substrates thus suggest that theaction of NPY is seizure- and circuit-specific, and verylikely depends on the activation of NPY receptors in brainstructures other than the hippocampus [3] and which areinvolved either in the initiation, propagation or control ofseizures [14].

4.3. Pharmacology of the effects of NPY on epilepticactivity

There are at present few selective and high affinity ago-nists and antagonists for NPY receptors [8,15,60]. This hashampered the determination of the receptors responsible forthe different biological effects of NPY. Therefore, pharma-cological studies on the modulation of hippocampal excit-ability by NPY have, up to now, been essentially based onthe use of semi-selective agonists with high affinity (lownanomolar range) for the Y1, Y2, Y5 receptors expressed inthe hippocampus [17,18,19,24,26,31,52,55]. In this study,we used human NPY, human [Leu31Pro34]PYY (whichbinds preferentially to Y1 . Y5), human PYY3-36 (Y2 .Y5 @ Y1 agonist), porcine NPY13-36 (Y2 @ Y5 @ Y1

agonist), and human PP (Y5 agonist) [6,24,48]. These arethe most commonly used agonists in pharmacological stud-ies of NPY biological effects [35,76]. The pharmacologicalprofile of the feeding behavior observed in our study fol-lowing ICV injection of these agonists, which is probably

secondary to the activation of Y1 and Y5 receptors withinthe hypothalamus, agrees with other descriptions and con-stitutes a control for the activity of the agonists used [33].

In our study, differing effects of the Y1-preferring ago-nist were observed. Treatment of hippocampal cell cultureswith the Y1/Y5 agonist reduced seizure-like activity. Thisinhibitory effect could result from Y1 activation, as thisreceptor is strongly expressed in cultured glutamatergichippocampal neurons as opposed to Y5 and Y2 [68,69]. Ithas been shown that, [Leu31Pro34]NPY also reduces epilep-tiform activity in hippocampal slices [2].In vivo, ICV in-jection of [Leu31Pro34]PYY attenuated the epileptiform re-sponse to electrical stimulation of the hippocampus. Thiseffect could be related to the reduction of intracellularcalcium influx which can be induced by Y1 activation andwhich may point to a mechanism of presynaptic inhibition[47]. On the contrary, intrahippocampal injection of[Leu31Pro34]PYY increased the duration of primary hip-pocampal afterdischarges, in accordance with recentin vivoevidence suggesting that activation of Y1 receptors is pro-convulsant [23]. These differences in the effects of Y1

activation in vitro and in vivo may depend on the modelused or the cell type studied. Moreover, the effects observedfollowing the use of a Y1/Y5 agonist may result from abalance between the excitatory and inhibitory effects result-ing from the activation of Y1 and Y5 receptors (see below),respectively.

In vitro, NPY, Y2/Y5 and Y5 agonists diminished epilep-tiform activity in cultures of hippocampal cells.In vivo,significant reductions of afterdischarge durations and wetdog shakes were obtained after intracerebral injection of theY2/Y5 and Y5 preferring agonists in the hippocampal stim-ulation paradigm. Taken together, these results suggest thatactivation of Y5 receptors or possibly of both Y2 and Y5

receptors can reduce hippocampal excitability. The absenceof a significant inhibitory effect of NPY applied ICV onsecondary afterdischarges may result from a balance be-tween the effects of Y1 (see above), Y2 and Y5 activation.Our results are in agreement with electrophysiological re-cordings from hippocampal slices, demonstrating that pre-synaptic activation of Y2 receptors, at the mossy fiber toCA3 and Schaffer collateral to CA1 synapses, decreasesfeedforward synaptic transmission, glutamate release andexperimentally induced epileptiform activity [5,30,35,46,54]. The role of Y5 receptors, mainly located in the CA3region and the dentate gyrus [17,26,55], is less consensual.Y5 activation in vivo inhibits limbic seizures and statusepilepticus elicited by kainic acid [76] and Y5-deficientmice are also more sensitive to these kind of generalizedseizures [43]. However, application of Y5 agonists ([Ahx8–

20][Pro34]NPY or D-Trp32NPY) on hippocampal slices doesnot suppress epileptiform activity in CA3, despite reducingsynaptic excitation [32].

Finally, in the model of generalized convulsive seizuresinduced by pentylenetetrazol, ICV injection of NPY, Y1 andY2/Y5 preferring agonists led to increased seizure duration.


The Y2 and Y5 preferring agonists were ineffective individ-ually, suggesting a possible additive effect when both re-ceptors are activated simultaneously. Further studies arenecessary to understand what brain structures are involvedin these proconvulsant properties and their pharmacology.

In conclusion, this study shows that NPY reduces hip-pocampal excitabilityin vitro and in vivo, an effect whichappears to be related at least in part to Y5 receptors activa-tion. On the contrary, excitatory effects of Y1 preferringagonists were observedin vivo. Concomitantly with theenhanced expression of NPY induced by seizures, bindingand expression of Y2 and Y5 receptors increase in thehippocampus in different models of epilepsy [27,28,37,57,62]. On the contrary, a reduction of hippocampal Y1 ex-pression is observed [36]. Taken together, these modifica-tions would be expected to promote the anticonvulsantproperties of NPY. Therefore, seizures appear to trigger inthe hippocampus a cascade of changes involving NPY andits receptors, which may counteract excessive excitation ofthis structure. However, these anticonvulsant properties ap-pear to be specific to seizures of hippocampal origin. Theproconvulsant effects observed in a model of generalizedclonic seizures suggest that NPY may not constitute a broadspectrum anticonvulsant.

Acknowledgments

This work was funded by INSERM, NATO (collabora-tive research grant n°961200) and Naval Medical Researchand Development Command work unit no. 61153NMR04101.001-1601. Sophie Reibel was supported by agrant from the Ministe`re de l’Enseignement Supe´rieur et dela Recherche (grant n°95094) and by the Fondation pour laRecherche Me´dicale. The authors thank Amgen Center forgenerously providing neuropeptide Y agonists.

The opinions and assertions contained herein are theprivate ones of the authors and are not to be construed asofficial or reflecting the views of the Navy Department orthe Naval Service at large.

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Neuropeptide Y and epilepsy: varying effects according to seizure type and receptor activation