modulation of intracellular calcium changes and glutamate release by neuropeptide y1 and y2...

Journal of Neurochemistry, 2001, 79, 286±296

Modulation of intracellular calcium changes and glutamate release

by neuropeptide Y1 and Y2 receptors in the rat hippocampus:

differential effects in CA1, CA3 and dentate gyrus

Ana P. Silva,* ArseÂlio P. Carvalho,* Caetana M. Carvalho* and JoaÄo O. Malva*,²

*Center for Neuroscience of Coimbra, Department of Zoology and ²Laboratory of Biochemistry, Faculty of Medicine, University of

Coimbra, Coimbra, Portugal

Abstract

In the present work, we investigated the role of pre- and

post-synaptic neuropeptide Y1 (NPY1) and Y2 receptors on

the calcium responses and on glutamate release in the rat

hippocampus. In cultured hippocampal neurones, we observed

that only NPY1 receptors are involved in the modulation of

intracellular free calcium concentration ([Ca21]i). In 88% of the

neurones analysed, the increase in the [Ca21]i, in response

to depolarization with 50 mM KCl, was inhibited by 1 mM

[Leu31,Pro34]NPY, whereas 300 nM NPY13±36 was without

effect. However, studies with hippocampal synaptosomes

showed that both NPY1 and Y2 receptors can modulate the

[Ca21]i and glutamate release. The pharmacological charac-

terization of the NPY-induced inhibition of glutamate release

indicated that Y2 receptors play a predominant role, both in the

modulation of Ca21-dependent and -independent glutamate

release. However, we could distinguish between Y1 and Y2

receptors by using [Leu31,Pro34]NPY and NPY13±36. Active

pre-synaptic Y1 receptors are present in the dentate gyrus

(DG) as well as in the CA3 subregion, but its activity was not

revealed by using the endogenous agonist, NPY. Concerning

the Y2 receptors, they are present in the three subregions

(CA1, CA3 and DG) and were activated by either NPY13±36

or NPY. The present data support a predominant role for

NPY2 receptors in mediating NPY-induced inhibition of gluta-

mate release in the hippocampus, but the physiological

relevance of the presently described DG and CA3 pre-

synaptic NPY1 receptors remains to be clari®ed.

Keywords: glutamate release, hippocampus, intracellular

calcium, Y1 receptors, Y2 receptors.

J. Neurochem. (2001) 79, 286±296.

Neuropeptide Y (NPY) is the most abundant peptide in the

mammalian central and peripheral nervous systems, exhibiting

potent effects on feeding, memory, blood pressure, cardiac

contractility and intestinal secretions (Balasubramaniam

1997). The use of various cloning techniques has resulted

in the identi®cation of ®ve receptors (Y1, Y2, Y4, Y5 and

Y6) (Wan and Benjamin 1995), and a more recent study

shows the existence of a putative Y3 receptor (Lee and

Miller 1998). The NPY receptors, especially Y1, Y2 and Y5,

are most abundant in the hippocampal formation (Aicher

et al. 1991; Dumont et al. 1993, 1998; Parker and Herzog

1999), where they modulate cognitive functions. NPY2

binding sites are particularly concentrated in the strata

radiatum and oriens of the CA1 sub®elds, and throughout

the pyramidal cell layer of the CA3 region (Parker and

Herzog 1999). In contrast, Y1 binding sites are mostly

localized in the granular layer of the dentate gyrus (DG;

Dumont et al. 1993, 1996; Larsen et al. 1993).

NPY receptors show very low primary amino acid

sequence identity, but surprisingly, they exhibit very similar

pharmacology. Indeed, it is possible to characterize the

receptors based on the rank order of potency of NPY and

related peptides (Michel et al. 1998) and of selective

antagonists (Lundberg et al. 1996). The Y1 subtype displays

high af®nity for the NPY analogue [Leu31,Pro34]NPY and

for the Y1 receptor antagonist BIBP3226 (Wieland et al.

286 q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 79, 286±296

Received April 9, 2001; revised manuscript received July 26, 2001;

accepted July 27, 2001.

Address correspondence and reprint requests to J. O. Malva, Center

for Neuroscience of Coimbra, Department of Zoology, University of

Coimbra, 3004±517 Coimbra, Portugal. E-mail: [email protected]

Abbreviations used: 4-AP, 4-aminopyridine; BIBP3226, (R)-N 2-

(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]-d-arginine amide; BSA,

bovine serum albumin; [Ca21]i, intracellular free calcium concentra-

tion; CA1, regio superior cornu ammonis; CA3, regio inferior cornu

ammonis; DG, dentate gyrus; NPY, neuropeptide Y.

1995; Doods et al. 1996). In contrast, the Y2 receptor

subtype exhibits high af®nity for C-terminal fragments of

NPY (Rose et al. 1995; Wieland et al. 1995), and also for

two different Y2 receptor antagonists, BIIE0246 (Doods

et al. 1999) and T4-[NPY(33±36)]4 (Grouzmann et al.

1997).

Although we do not know the precise role of each NPY

receptor subtype in the various physiological effects induced

by NPY, the Y1 receptor has been linked to different

biological actions, in the central nervous system, such as

NPY-induced stimulation of feeding behaviour (Wieland

et al. 1998) or the modulation of anxiety (Wahlestedt et al.

1993). The Y2 receptors are predominantly known for their

association with inhibitory effects on the release of gluta-

mate in the rat hippocampus, and for their effects on the

modulation of memory processes. Also, NPY is a powerful

stimulant for food intake by activating the hypothalamic

`feeding' receptor, the NPY5 receptor.

Since NPY inhibits excitatory neurotransmission and

regulates hyperexcitability (Baraban et al. 1997) in normal

hippocampus (Colmers et al. 1991), this peptide may play a

critical role during seizure activity (Woldbye et al. 1997).

However, the NPY receptor subtype(s) mediating endo-

genous anticonvulsant effects has not been clearly de®ned,

but it seems that Y1, Y2 and Y5 subtypes are likely to play

an important role in normal physiological conditions, as well

as in response to pathological hyperactivity in the hippo-

campus (Bijak 1999; Marsh et al. 1999). Indeed, the pre-

synaptic modulation of glutamate release by NPY receptors

may be especially relevant for antiepileptic properties of

NPY. Recently, a pre-synaptic Y2 receptor in CA1 sub-

region was described (Weiser et al. 2000). However, there is

a lack of pharmacological characterization of NPY1 and Y2

pre- and post-synaptic receptors in the hippocampal sub-

regions. The present paper aims to clarify the contribution of

both receptors in modulating the intracellular calcium

changes and glutamate release in the hippocampus. Pre-

liminary results of this work were previously published in

abstract form (Silva et al. 2000).

Materials and methods

Materials

Neuropeptide Y, neuropetide Y13±36 and [Leu31,Pro34]neuro-

petide Y were obtained from Bachem (Bubendorf, Switzerland).

(R)-N 2-(Diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]arginin amide

(BIBP3226) was purchased from Peninsula Laboratories (Belmont,

CA, USA), and T4-[neuropeptide Y(33±36)]4 was provided by Dr

Eric Grouzmann. Neurobasal medium, B27 supplement, gentamicin

and trypsin (USP grade) were purchased from Gibco-BRL, Life

Technologies (Paisley, UK). Glutamate, DNase (DN-25) and

l-glutamic acid dehydrogenase type II were obtained from Sigma

Chemical (St Louis, MO, USA). The acetoxymethyl ester of fura-2

(fura-2AM) and Pluronic F-127 were purchased from Molecular

Probes (Leiden, the Netherlands). All the other reagents were from

Sigma or from Merck-Schuchardt (Darmstadt, Germany). Fura-

2AM and Pluronic F-127 stock solutions were prepared in

dimethylsulfoxide (DMSO).

Cell culture

Hippocampal neurones were dissociated from hippocampi of E18-

E19 Wistar rat embryos, after treatment with trypsin (2.0 mg/mL,

15 min, 378C) and deoxyribonuclease I (0.15 mg/mL) in Ca21 and

Mg21 free Hank's balanced salt solution (137 mm NaCl, 5.36 mm

KCl, 0.44 mm KH2PO4, 0.34 mm Na2HPO4.2H2O, 4.16 mm

NaHCO3, 5 mm glucose, 1 mm sodium pyruvate, 10 mm HEPES,

pH 7.4). The cells were cultured in serum-free neurobasal medium,

supplemented with B27 supplement, glutamate (25 mm), glutamine

(0.5 mm) and gentamicin (0.12 mg/mL), as described previously

(Brewer et al. 1993; AmbroÂsio et al. 2000). Cultures were kept at

378C in a humidi®ed incubator in 5% CO2/95% air, for 7±8 days, the

time required for maturation of hippocampal neurones. For calcium

imaging, cells were plated on poly-d-lysine-coated (0.1 mg/mL)

glass coverslips at a density of 45 � 103 cells/cm2.

Procedures involving animals and their care were carried out in

accordance with European Community laws and policies. All

efforts were made to minimize animal suffering, and to reduce the

number of animals used.

Preparation of synaptosomes

A partially puri®ed synaptosomal fraction (P2) was isolated from

hippocampi or from hippocampal subregions (CA1, CA3 and DG)

of male Wistar rats (2-month-old), essentially as described pre-

viously for brain cortex (McMahon et al. 1992), with some modi®-

cations (Malva et al. 1996). The hippocampi were homogenized in

0.32 m sucrose, 10 mm HEPES-Na, pH 7.4, and centrifuged at

3000 g for 2 min. The pellet obtained was resuspended, followed

by sedimentation at the same speed. The combined supernatants

were spun for 12 min at 14 600 g, and a P2 pellet was obtained.

The upper, whiter, layer of this pellet was resuspended in the

sucrose medium.

Coronal slices of the hippocampus (800 mm thick) were prepared

for the isolation of synaptosomes from hippocampal subregions

(CA1, CA3 and DG). In each slice, the ®mbria and the subiculum

were separated from the rest of each slice, under stereomicroscopic

observation. CA3 subslices were obtained by separation from CA1

and DG, and the last separation (CA1 from DG) was performed

through the hippocampal sulcus (Fig. 1). The pooled subslices were

homogenized in the sucrose medium indicated above, transferred to

Eppendorf tubes and centrifuged as described for the isolation of

whole hippocampal synaptosomes. The P2 pellet of each subregion

was resuspended in buffered sucrose medium (Malva et al. 1996).

For the [Ca21]i measurements or glutamate release, the synapto-

somes were stored as drained pellets, containing 0.75 mg or 1 mg

of protein each, respectively.

Percoll-puri®ed synaptosomes were also prepared (Dunkley et al.

1988), for comparison, and we obtained similar results in the

intracellular calcium changes and glutamate release protocols.

[Ca21]i measurements in synaptosomal preparations

Synaptosomes (3 mg/mL) were incubated with 5 mm fura-2AM

and 0.02% Pluronic F-127 in incubation medium (132 mm NaCl,

1 mm KCl, 1 mm MgCl2, 100 mm CaCl2, 1.2 mm H3PO4, 10 mm

glucose and 10 mm HEPES-Na, pH 7.4) with 0.1% fatty acid-free

bovine serum albumin (BSA) for 20 min at 258C. After this loading

Neuropeptide Y1 and Y2 receptors in the hippocampus 287

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 79, 286±296

period, synaptosomes were pelleted, the non-hydrolysed probe was

removed and further incubated in the same medium plus 1 mm

[Leu31,Pro34]NPY or 300 nm NPY13±36, for 10 min at 378C.

Synaptosomes were also preincubated with the agonists for 300 s

before stimulation with 1 mm 4-aminopyridine (4-AP). The

¯uorescence of fura-2-loaded synaptosomes was monitored at

378C, using a computer-assisted Spex Fluoromax spectro¯uoro-

meter, at 510 nm emission and double excitation at 340 nm and

380 nm, using 5 nm slits. The calibration was made in the presence

of 2.5 mm ionomycin (1 mm CaCl2; Rmax), at 500 s, and 4 mm

EGTA (Rmin), at 600 s. The ¯uorescence intensities were auto-

matically converted into [Ca21]i-values by using the calibration

equation for double excitation wavelength measurements and

taking the dissociation constant of the fura-2/Ca21 complex as

224 nm (Grynkiewicz et al. 1985).

Fura-2 ¯uorescence measurements by video imaging in

hippocampal neurones

Hippocampal neurones plated on coverslips at a density of

45 � 103 cells/cm2 were loaded by incubation with 5 mm fura-

2AM and 0.02% Pluronic F-127 for 45 min, at 378C, in Krebs

buffer supplemented with 0.1% BSA (w/v). After incubation, the

coverslips were washed and placed in a perfusion chamber on

the stage of an inverted Nikon Diaphot ¯uorescence microscope.

The cells were then perfused with Krebs buffer (378C) for about

5 min, before data acquisition. The experiments were conducted

under continuous superfusion with Krebs buffer containing the

drugs, as indicated in the ®gure legends. The neurones were

stimulated with 50 mm KCl, 15 s after starting image acquisition

and preincubated with [Leu31,Pro34]NPY (1 mm) or NPY13±36

(300 nm) for 10 min before stimulation. The ¯uorescence changes

were recorded with a multiple excitation Magical imaging system

(Applied Imaging, Sunderland, UK). Hippocampal neurones were

alternately excited at 340 nm and 380 nm using a switching ®lter

wheel, and the emitted ¯uorescence, collected with a 40� objective

(Nikon, Badhoeverdoer, the Netherlands), was driven to a

Photonics Science SIT camera, after passing through a 510-nm

bandpass ®lter. Image analysis was performed with the Magical

system and software developed by Dr Enrique Castro (Department

of Biochemistry and Molecular Biology and Physiology, University

Las Palmas, Gran Canaria, Spain). The ratio images were coded in

pseudocolour (dark blue to red for minimum to the maximum 340/

380 ratios), and the background ¯uorescence at each wavelength

was subtracted and ¯uorescence images were ratioed on a pixel-by-

pixel basis, being ratio data stored as a 8-bit pseudocoloured

images. Areas of the cell bodies were drawn and the averaged value

of pixel intensities was evaluated at each time point, in order to

obtain ratio versus time plots for all areas de®ned. The results were

expressed as the ratio of ¯uorescence intensity with excitation at

340 nm and 380 nm.

Measurement of glutamate release

The release of endogenous glutamate was followed using a con-

tinuous ¯uorimetric assay as previously described (Nicholls et al.

1987), with some modi®cations (Malva et al. 1996). Synaptosomes

(1 mg protein) were incubated for 20 min at 378C in the following

medium: 132 mm NaCl, 1 mm KCl, 1 mm MgCl2, 1.2 mm H3PO4,

0.1 mm CaCl2, 10 mm glucose, 10 mm HEPES-Na, pH 7.4, with

0.1% fatty acid-free BSA. After this period, the agonists or/and

antagonists were added to the medium for 10 min Then, synapto-

somes were centrifuged at 15 800 g, and resuspended in 1 mL of

the same medium, without BSA and with 1 mm CaCl2 or 200 nm

free Ca21. The suspension was transferred to a stirred cuvette at

378C, followed by the addition of 1 mm NADP, 50 units of puri®ed

glutamate dehydrogenase, and again the agonists/antagonists. The

total period of exposure to the drugs was 20 min. Fluorescence was

measured by using a Perkin-Elmer model LS-5B luminescence

spectrometer at the excitation and emission wavelengths of 340 nm

and 460 nm, respectively, with excitation and emission slits of

5 nm and 10 nm, respectively. The data were collected at 0.5-s

intervals and the quanti®cation of glutamate was performed at the

end of each experiment by adding 2.5 nmol of l-glutamate.

Statistical analysis

The data are expressed as means ^ SEM. Statistics were performed

using an analysis of variance (anova), followed by Dunnett's or

Bonferroni's post-tests, as indicated in the ®gure legends. In the

[Ca21]i measurements, the 95% con®dence intervals (CI) are also

indicated. The dose±inhibition curves for the effects of [Leu31,-

Pro34]NPY or NPY13±36 represent the best ®t according the non-

linear regression analysis (one-site competition), assuming top

value of 100%.

Fig. 1 Coronal section (800 mm) of the hippocampus obtained from

a 6-week-old male Wistar rat, and illustration of the procedure used

to visualize the subregions before isolation of the synaptosomal frac-

tion. For the illustration, the slice was ®xed in 4% paraformaldehyde,

followed by Timm staining. (a) Whole slice. (b) Separation of CA3

from the ®mbria plus CA1/DG subregions, and separation of DG

from CA1 through the hippocampal ®ssure. CA1, regio superior

cornu ammonis; CA3, regio inferior cornu ammonis; DG, dentate

gyrus.

288 A. P. Silva et al.


Results

Modulation of the [Ca21]i response by NPY1 and Y2

receptor activation

The in¯ux of calcium is a key signal for cell death, and a

prolonged increase in intracellular Ca21 concentration is

regarded as one major trigger of the neurodegenerative

processes. To better understand the possible modulatory role

of NPY1 and Y2 receptors in neuroprotection, we analysed the

[Ca21]i changes evoked by KCl depolarization in the presence

of selective agonists for these receptors in cultured hippo-

campal neurones, as well as in hippocampal synaptosomes.

In single cell studies, we observed that, in most cells

analysed, the [Ca21]i responses induced by 50 mm KCl were

inhibited by 1 mm [Leu31,Pro34]NPY (Figs 2a±d and 3a±c),

but not by 300 nm NPY13±36 (Figs 2e±h and 3d), two

agonists of either NPY1 or Y2 receptors, respectively. How-

ever, different populations of neurones responded differently

to NPY1 and Y2 receptors activation: the presence of 1 mm

[Leu31,Pro34]NPY signi®cantly reduced the KCl-stimulated

[Ca21]i changes in 89% of total neurones (144 neurones

analysed), and in a smaller percentage of neurones (11%)

this Y1 receptor agonist did not have any effect (Fig. 3a,

group A). Concerning the inhibitory effect induced by

[Leu31,Pro34]NPY, we obtained two groups of neurones:

group B (Fig. 3b), including neurones in which the KCl-

evoked response in the presence of [Leu31,Pro34]NPY was

between 10 and 50% of the control; and group C (Fig. 3c)

including neurones in which the response was between 50

and 90% of the control. Thus, in group B (24.3% of total

neurones), the response was inhibited to 32.2 ^ 1.9% (95%

CI between 12.0 and 48.9%), whereas in group C (64.6% of

total neurones), the response was inhibited to 67.4 ^ 1.0%

of the control (95% CI between 51.1 and 88.8%). In contrast,

incubation with the NPY2 receptor agonist, NPY13±36

(300 nm), did not signi®cantly modulate the [Ca21]i in

neuronal cell bodies, in all cells analysed (Fig. 3d).

In order to investigate pre-synaptic modulation of [Ca21]i

by NPY receptors we used rat hippocampal synaptosomes.

Fura-2 loaded synaptosomes were depolarized with KCl

(15 mm), resulting in a subsequent increase in [Ca21]i. In

the presence of [Leu31,Pro34]NPY (1 mm) or NPY13±36

(300 nm), the increase in [Ca21]i due to KCl stimulation was

inhibited to 86.0 ^ 1.6% or 79.6 ^ 2.1% of the control,

respectively (Fig. 4). Similar inhibitory effects were

obtained when using 1 mm 4-aminopyridine (4-AP), instead

of 15 mm KCl as the depolarizing agent (data not shown).

Modulation of endogenous glutamate release by

activation of NPY1 and Y2 receptors in hippocampal

nerve terminals: effects on Ca21-dependent and

Ca21-independent release

Glutamate is the major excitatory neurotransmitter in the

mammalian CNS and is known to be involved in some CNS

pathologies. Due to their putative antiepileptic and neuro-

protective properties, it is important to understand how NPY

receptors can regulate glutamate release. Therefore, we

investigated the effect of different concentrations of

[Leu31,Pro34]NPY or NPY13±36 on the release of

endogenous glutamate stimulated by KCl (15 mm).

The two agonists inhibited both the total (1 mm Ca21

present) and the Ca21-independent (200 nm free external

Ca21) glutamate release in a concentration-dependent

manner (Figs 5a and b). However, although the maximal

inhibition of Ca21-independent release of glutamate was

Fig. 2 Fluorescence microscopy images of single-cell Ca21 concen-

tration in monolayer cultures of hippocampal neurones. (a, c, e and

g) The ®gures represent the basal [Ca21]i; (b and f ) 15 s after stimu-

lation with 50 mM KCl; 15 s after stimulation with 50 mM KCl in the

presence of (d) 1 mM [Leu31,Pro34]NPY or (h) 300 nM NPY13±36.

The images were coded in pseudocolour (dark blue to red for the

minimum to the maximum 340 nm/380 nm ratios).



similar in both situations, at the same concentration of the

agonists (the bottom values were 60.7% or 70.7%, for

[Leu31,Pro34]NPY or NPY13±36, respectively), the NPY2

receptor activation induced a higher inhibition of the total

glutamate release, when compared with the activation of Y1

receptors (the bottom values were 67.5 or 49.5, for

[Leu31,Pro34]NPY or NPY13±36, respectively). In the

presence of [Leu31,Pro34]NPY, the calculated IC50 values

to the glutamate release inhibition were 1092 nm (95% CI

between 424 and 2815) or 124 nm (95% CI between 68 and

224), with 200 nm or 1 mm Ca21 present in the medium,

respectively (Fig. 5a). Moreover, in the presence of NPY13±

36, the IC50s values were 81 nm (95% CI between 44 and

151) or 99 nm (95% CI between 62 and 159), when 200 nm

or 1 mm Ca21 was present in the medium, respectively

(Fig. 5b). Based on these ®ndings, we used selected con-

centrations of the two agonists (1 mm [Leu31,Pro34]NPY,

100 or 300 nm NPY13±36) to further characterize the

effects of NPY1 and Y2 receptor activation on the Ca21-

dependent and -independent glutamate release, as shown in

Figs 6 and 7.

Stimulation of hippocampal synaptosomes with 15 mm KCl

(1 mm Ca21 present) evoked the release of 1.29 ^ 0.11%

nmol glutamate/mg protein/min. The Ca21-independent

glutamate release, presumably due to the reversal of glutamate

carrier, obtained in the presence of 200 nm free external

Ca21, was 0.39 ^ 0.03% nmol glutamate/mg protein/min,

which corresponds to 30% of the total glutamate release. In

the presence of 1 mm [Leu31,Pro34]NPY, 100 or 300 nm

NPY13±36 or 1 mm NPY, the total glutamate release

decreased to 71.6 ^ 1.8%, 71.3 ^ 2.0%, 60.1 ^ 2.9% or

48.2 ^ 2.2% of the control, respectively (Fig. 6a).

In order to show the selective involvement of the NPY

receptors on total glutamate release, we also used NPY1 and

Y2 receptor antagonists. The selective Y1 receptor antago-

nist, BIBP3226 (1 mm), completely prevented the inhibitory

effect induced by 1 mm [Leu31,Pro34]NPY (93.4 ^ 2.3%

Fig. 3 Representative recordings of single-

cell [Ca21]i responses to 50 mM KCl depo-

larization, in the absence or in the presence

of (a±c) [Leu31,Pro34]NPY (1 mM) or (d)

NPY13±36 (300 nM). Concerning the effect

of [Leu31,Pro34]NPY, three groups of neu-

rones were identi®ed showing: (a) no inhibi-

tion, (b) strong or (c) moderate inhibition of

the [Ca21]i responses induced by 50 mM

KCl. The results were obtained from 144 or

50 cultured hippocampal neurones, respec-

tively (®ve or three independent cultures,

respectively). Hippocampal neurones were

stimulated 15 s after starting data acquisi-

tion and each recording lasted 75 s. The

second recording in each experiment was

carried out 15 min after the end of the ®rst

recording.

Fig. 4 Quantitative analysis of the inhibitory effect of [Leu31,

Pro34]NPY (1 mM) or NPY13±36 (300 nM) on the [Ca21]i changes sti-

mulated by 15 mM KCl in rat hippocampal synatosomes. The basal

[Ca21]i was 213.9 ^ 9.3 nM (n � 12). The results represent the

mean ^ SEM between four and eight independent experiments, in

different synaptosomal preparations. *p , 0.05 ± Dunnett's post hoc

test; statistical signi®cance when compared with the control (KCl

stimulation).



of the control) (Fig. 6a). Concerning the Y2 receptor, the

antagonist, T4-[NPY(33±36)]4 (1 mm), completely pre-

vented the inhibitory effect induced by 100 nm NPY13±36

(95.6 ^ 5.6% of the control), but at a higher concentration

of the agonist (300 nm), the blockade by the Y2 antagonist

Fig. 5 Concentration±inhibition curve for the effect of (a) [Leu31,

Pro34]NPY or (b) NPY13±36 10±3000 nM on the total (D) and

Ca21-independent (B) release of endogenous glutamate evoked by

15 mM KCl, determined in the presence of 1 mM or 200 nM CaCl2,

respectively. The results represent the mean ^ SEM of between

three and 12 independent experiments, in different synaptosomal

preparations. *p , 0.05, **p , 0.01 ± Dunnett's post hoc test;

Statistical signi®cance when compared with the control (KCl

stimulation in the absence of the agonists).

Fig. 6 Quantitative analysis of the effect of NPY1 and Y2 receptors

agonists and antagonists on the (a) total (1 mM Ca21 present in the

external medium), (b) Ca21-independent 200 nM free Ca21 in the

external medium and (c) Ca21-dependent glutamate release (differ-

ence between total and Ca21-independent glutamate release)

evoked by 15 mM KCl depolarization in hippocampal synaptosomes.

The results represent the mean ^ SEM of between three and seven

independent experiments, in different synaptosomal preparations.

*p , 0.05, **p , 0.01 ± Dunnett's post-test; Statistical signi®cance

when compared with the control (KCl stimulation).



was not complete (71.4 ^ 2.7% of the control) (Fig. 6a).

The inhibitory effect of NPY (1 mm) (48.2 ^ 2.2% of the

control) could not be completely blocked by the Y2 receptor

antagonist (73.8 ^ 3.7% of the control), nor was it altered

by the Y1 receptor antagonist (52.3 ^ 3.7% of the control)

(Fig. 6a). Also, in the presence of both antagonists, the

effect was similar to that obtained in the presence of

T4-[NPY(33±36)]4 only (76.3 ^ 1.6% of the control)

(Fig. 6a).

We also investigated the effect of the activation of Y1 and

Y2 receptors on the Ca21-independent glutamate release

evoked by 15 mm KCl, determined in the presence of

200 nm CaCl2. In the presence of 1 mm [Leu31,Pro34]NPY,

100 or 300 nm NPY13±36, the Ca21-independent glutamate

release decreased to 78.3 ^ 1.3%, 81.3 ^ 1.3% or 76.3 ^

1.0% (Fig. 6b) of the control, respectively. The inhibition of

Ca21-independent glutamate release induced by NPY

(1 mm) was similar to that obtained with [Leu31,Pro34]NPY

or NPY13±36 (79.2 ^ 3.5% of the control) (Fig. 6b).

Moreover, the veratridine-evoked Ca21-independent gluta-

mate release was inhibited in a very similar way by the

agonists (not shown).

The selective Y1 receptor antagonist, BIBP3226 (1 mm),

completely prevented the inhibitory effect due to 1 mm

[Leu31,Pro34]NPY (101.0 ^ 2.9% of the control) (Fig. 6b),

and the Y2 receptor antagonist, T4-[NPY(33±36)]4 (1 mm),

completely prevented the inhibitory effect due to 100 or

300 nm NPY13±36 (101.8 ^ 2.1% or 96.5 ^ 2.1% of the

control, respectively) (Fig. 6b). Also, it is interesting to

observe that the inhibitory effect of NPY on the Ca21-

independent glutamate release was mostly mediated by

NPY2 receptor activation, since the antagonist of Y2

receptors, T4-[NPY(33±36)]4, completely blocked the

inhibitory effect (100.0 ^ 2.1% of the control), which was

not the case in the presence of BIBP3226 (76.0 ^ 3.0% of

the control) (Fig. 6b). Similar results were obtained for the

effects of the antagonists on the total glutamate release

(Fig. 6a).

Based on the data presented in Fig. 6(a and b), it was

possible to calculate the effect of NPY1 and Y2 receptors

activation on the Ca21-dependent glutamate release (differ-

ence between total and Ca21-independent glutamate

release). In the presence of 1 mm [Leu31,Pro34]NPY, 100

or 300 nm NPY13±36 or 1 mm NPY, the Ca21-dependent

glutamate release decreased to 70.0 ^ 2.5%, 67.4 ^ 1.1%,

56.4 ^ 2.3% or 39.4 ^ 3.6% of the control, respectively

(Fig. 6c).

Modulation of glutamate release by activation of NPY1

and Y2 receptors in the hippocampal subregions: CA1,

CA3 and DG

Previous work indicates that the density of NPY1 and Y2

receptors varies among hippocampal subregions (Dumont

et al. 1993), and that the pre-synaptic effects of NPY in the

CA1 subregion are mediated by Y2 receptors (Weiser et al.

2000). To better characterize the nature of pre-synaptic NPY

receptors in different hippocampal subregions, we compared

the modulatory effect of [Leu31,Pro34]NPY and NPY13±36

on the glutamate release obtained in the whole hippocampal

synaptosomes, or in synaptosomes isolated from hippo-

campal CA1, CA3 or DG subregions. When synaptosomes

isolated from the whole hippocampus, from CA3 or

from DG subregions (Fig. 1), were stimulated with 15 mm

KCl, the total release of glutamate was inhibited by

[Leu31,Pro34]NPY (1 mm) to 71.6 ^ 1.8%, 51.3 ^ 3.6, or

60.0 ^ 4.3% of the control, respectively (Fig. 7a). How-

ever, in synaptosomes from the CA1 subregion, the NPY1

receptor agonist did not show any inhibitory effect

(103.2 ^ 3.4% of the control), and the inhibition obtained

in CA3 subregion was signi®cantly greater than that obtained

in the whole hippocampus (�p , 0.05). Moreover, the total

release of glutamate was also inhibited by NPY13±36

(300 nm) to 60.1 ^ 2.1%, 74.3 ^ 2.6%, 53.8 ^ 2.2%, or

63.0 ^ 3.9% of the control, in synaptosomes from the

whole hippocampus or from the CA1, CA3 or DG sub-

regions, respectively (Fig. 7a).

The Ca21-independent glutamate release was slightly

inhibited by the two agonists in the CA3 and DG subregions,

but in the CA1 subregion only the activation of the Y2

receptors induced an inhibition of glutamate release:

80.1 ^ 1.7% or 82.7 ^ 5.5% of the control, respectively,

for [Leu31,Pro34]NPY, and 81.0 ^ 1.1%, 77.3 ^ 1.1% or

78.0 ^ 2.3 of the control, in CA1, CA3 or DG, respectively,

for NPY13±36 (Fig. 7b). Therefore, the Ca21-dependent

glutamate release was greatly inhibited by the NPY1 and

Y2 receptor agonists, especially in the CA3 subregion

(46.3 ^ 6.8% or 41.7 ^ 10.8% of the control, respectively)

(Fig. 7c), but also with a very signi®cant effect in the DG

subregion (59.0 ^ 6.7% or 67.3 ^ 1.5% of the control,

respectively). However, the activation of NPY2 receptors in

the CA1 subregion also induced a strong inhibition of the

Ca21-dependent glutamate release (46.8 ^ 5.3% of the

control). Thus, it is likely that the inhibition of glutamate

release observed in the whole hippocampus nerve terminals

is mainly due to the activation of both receptors localized in

the CA3 and DG subregions, and also to the activation of Y2

receptors in the CA1 subregion.

Discussion

The major ®nding of the present study is the identi®cation

of Y2 receptors responsible for the NPY-induced inhibition

of glutamate release in the hippocampus. Moreover, the

use of [Leu31,Pro34]NPY and BIBP3226 could also reveal

the involvement of pre-synaptic Y1 receptors able to modu-

late glutamate release in dentate gyrus and CA3 subregions.

Concerning the CA1 subregion, only Y2 receptors are

involved in the modulation of glutamate release since it is



inhibited in the presence of NPY13±36 but is insensitive to

the Y1 receptor activation. We could also show that cultured

hippocampal neurones express post-synaptic Y1 receptors

involved in the inhibition of KCl-evoked increase in the

[Ca21]i.

The present work provides the ®rst cell-to-cell character-

ization of the role of NPY1 and Y2 receptors on the [Ca21]i

changes in the cultured rat hippocampal neurones. The

pharmacological identi®cation of Y1 receptors in single-cell

Ca21 imaging studies is evident since the presence of

[Leu31,Pro34]NPY signi®cantly reduced the KCl-induced

[Ca21]i changes in a high percentage of neurones. Also, we

found that there is a cell-to-cell variability, in that the

neurones did not respond equally to the NPY1 receptor

agonist, probably indicating that there is an heterogeneous

distribution of these receptors in the hippocampal neurone

cell bodies and/or the receptors may have different af®nities

for the agonist. However, the NPY2 receptor activation

did not modulate the [Ca21]i in neuronal cell bodies, in

contrast to the ®nding on synaptosomes. In this case, both

[Leu31,Pro34]NPY and NPY13±36 inhibited the changes in

intracellular Ca21 stimulated by KCl depolarization to a

similar extent.

It was reported previously that cultured hippocampal

neurones preferential express NPY1 over the Y2 receptor

subtype (St-Pierre et al. 1998), and also that Y1 receptors

exist on NPYergic hippocampal neurones (St-Pierre et al.

2000). Since hippocampal neurones in culture coexpress

active Y1 and Y2 receptors subtype, our results suggest that

in the hippocampus NPY2 receptors act mainly at pre-

synaptic nerve terminals, while Y1 receptors act both at pre-

and post-synaptic membranes, and that both receptors can

modulate the Ca21 in¯ux. This is also in agreement with

other studies where NPY was demonstrated to inhibit N-type

Ca21 channels through Y2 receptor activation (McCullough

et al. 1998), in NGF-differentiated PC12 cells, as well as

through Y1 receptors in rat dentate granule cells (McQuiston

and Colmers 1996).

It has been postulated that pre-synaptic inhibition of

transmitter release by neuropeptide Y is due to a reduction

in Ca21 in¯ux at the nerve terminal (Toth et al. 1993). In

the present work, we have clearly demonstrated that the

pre-synaptic inhibition of intracellular Ca21 changes upon

selective activation of either NPY1 or Y2 receptors, is

coupled to the inhibition of endogenous glutamate release.

This is in agreement with the suggestion that NPY can act as

an endogenous neuromodulator of the glutamatergic neuro-

transmission, and also with the observation that NPY limits

hyperexcitability in the epileptic human DG (Patrylo et al.

Fig. 7 Quantitative analysis of the effect of [Leu31,Pro34]NPY

(1 mM) or NPY13±36 (300 nM) on the (a) total, (b) Ca21-independent

and (c) Ca21-dependent glutamate release evoked by 15 mM KCl in

synaptosomes obtained from the whole hippocampus and from the

subregions, CA1, CA3 and dentate gyrus (DG). The results repre-

sent the mean ^ SEM of between three and 12 independent experi-

ments, in different synaptosomal preparations. *p , 0.05; **p , 0.01

± Dunnett's post hoc test; statistical signi®cance when compared

with the control (KCl stimulation). �p , 0.05 ± Bonferroni's post-

test; Statistical signi®cance when compared with the whole hippo-

campus.



1999). NPY was also shown to inhibit glutamate release

through Y2 receptors in rat hippocampal slices in vitro

(Greber et al. 1994), and the involvement of Y1 receptors in

the pre-synaptic modulation of glutamate release in the

striatum was also reported (Ellis and Davies 1994).

In the present study we attempted to discriminate between

the effects of activating NPY1 or Y2 receptors on the two

components of endogenous glutamate release: the Ca21-

dependent and -independent components which may be

elicited simultaneously due to depolarization of the nerve

terminals (Nicholls et al. 1987). A new ®nding of the present

work is that both Y1 and Y2 receptors modulate (inhibit) not

only the Ca21-dependent exocitosis of glutamate, but

also the Ca21-independent non-vesicular pathway. Con-

cerning the inhibition of the Ca21-independent glutamate

release, the modulatory effects of both receptors may be

exerted through the ion channels involved in depolarization,

and/or the direct inhibition of glutamate carrier. It is known

that under certain conditions, the uptake carriers working in

reverse order can pump transmitter out of the cell, and serve

as a calcium-independent, non-vesicular mechanism for

transmitter release (Attwell et al. 1993; Nicholls 1993).

Another possible mechanism by which NPY can produce

pre-synaptic inhibition was shown in the arcuate nucleus,

involving Y1 receptor-mediated activation of K1 currents

(Rhim et al. 1997). Also, the activation of the Y2 receptor

can lead, through a pertussis toxin-sensitive G protein, to

the opening of inwardly rectifying potassium channels in

the Xenopus oocytes (Rimland et al. 1996). Other studies

showing the effects of NPY on pre-synaptic inhibition

induced by 4-aminopyridine (4-AP), suggest that NPY

causes pre-synaptic inhibition by increasing K1 conduc-

tance at the pre-synaptic terminal in rat hippocampal slices

in vitro (Klapstein and Colmers 1992). In hippocampal

neurones, the resulting increase of pre-synaptic K1 currents

by the activation of Y1 or Y2 receptors may inhibit the KCl-

evoked glutamate release.

We also determined that the total glutamate release

inhibition by NPY is mediated by NPY2 receptor activation,

and not by Y1 receptors. However, the NPY2 receptor

antagonist did not completely prevent this effect. This

®nding may suggest that receptors other than Y2 receptors,

for instance NPY5 receptors, may be involved in the

modulation of glutamate release by NPY. Indeed, Y5

receptors are expressed in high levels in the hippocampus

(Dumont et al. 1998), and mediate pre-synaptic inhibition of

stratum radiatum-evoked glutamatergic responses in some

pyramidal neurones of area CA3 and the proximal subicu-

lum (Ho et al. 2000). However, since T4-[NPY(33±36)]4 is a

competitive antagonist, it is also possible that this con-

centration (1 mm) may not be suf®ciently high to block fully

putative pre-synaptic Y2 receptors activated with 300 nm

NPY13±36. In fact, a recent study showed that the activa-

tion of Y2 receptors could inhibit basal and stimulated NPY

release, but this effect could only be partially prevented by

this Y2 antagonist (King et al. 1999). A more selective Y2

receptor antagonist, BIIE0246, with af®nity approximately

100-fold higher than T4-[NPY(33±36)]4, has recently been

synthesized (Doods et al. 1999), and can be useful in

clarifying these effects in our model.

We have shown that the activation of NPY1 and Y2

receptors inhibited glutamate release in the whole hippo-

campus, as well as in hippocampal subregions. These results

are in the line of previous work showing that NPY can

inhibit epileptiform activity in the rat hippocampus in vitro,

which can be due to inhibition of glutamate-mediated

synaptic transmission in areas CA1 and CA3 (Klapstein and

Colmers 1997), or in epileptic humans DG (Patrylo et al.

1999). The activation of post-synaptic Y1 receptors has a

depolarizing action on granule neurones when applied to

their dendritic projection in the stratum moleculare (Brooks

et al. 1987). Moreover, NPY acting on NPY2 receptors

inhibits excitatory (glutamatergic) synaptic transmission

(Colmers et al. 1991) onto CA3 pyramidal cells (McQuiston

and Colmers 1996). Recently a pre-synaptic Y2 receptor

was also identi®ed as the NPY receptor responsible for the

NPY-mediated inhibition of glutamate release in the CA1

subregion (Weiser et al. 2000).

In the present study, by using nerve terminals isolated

from the hippocampal subregions, CA1, CA3 and DG, we

show that the inhibition of Ca21-dependent and -indepen-

dent glutamate release is mainly due to the activation of Y1

receptors present in the CA3 and DG subregions, and to the

activation of Y2 receptors present in CA1, CA3 and DG

hippocampal subregions. However, in spite of the lower

expression of these receptors in the DG when compared

with the other two subregions (Parker and Herzog 1999),

we observed a clear inhibitory effect in this hippocampus

subregion. Accordingly, previous studies also showed that

NPY inhibits glutamate release (glutamatergic excitation) in

the rat DG (Whittaker et al. 1999), or even in epileptic

human DG (Patrylo et al. 1999). Moreover, the lack of effect

of [Leu31,Pro34]NPY in the CA1 subregion does not impli-

cate the absence of Y1 receptors, but only that when

selectively activated they do not modulate the glutamate

release in this hippocampal subregion. Thus, it is important

to keep in mind that the distribution of mRNA for NPY

receptors is not always indicative of the distribution of the

expressed proteins at synaptic levels. Indeed, for instance,

there is a lack of correlation between Y5 mRNA levels and

binding sites in the human hypothalamus (Statnick et al.

1998).

In conclusion, the data reported in the present paper show

that the activation of pre-synaptic NPY1 and Y2 receptor

can inhibit [Ca21]i changes and glutamate release in hippo-

campal nerve terminals. However, only NPY1 receptors

clearly modulate intracellular Ca21 changes in hippocampal

neurones. The NPY-mediated inhibition of glutamate



release in the hippocampus is due to the activation of Y2

receptors, but active pre-synaptic Y1 receptors were

revealed in the CA3 and DG subregions.

Acknowledgements

We are grateful to Dr GracËa Baltazar and to Dr Paulo Pinheiro

for helping us in ratio images analysis and in slice preparation,

respectively. We would also like to thank to Ms Elisabete

Carvalho for assisting us in hippocampal cell cultures. This

work was supported by Foundation for Science and Tech-

nology, PRAXIS XXI Program, Portugal (Project PRAXIS

XXI/35875/99 and Grant PRAXIS 18261/98).

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