neuropeptide y receptors in rat brain: autoradiographic localization

Peptides, Vol. 7, pp. 55-60, 1986. © Ankho International Inc. Printed in the U.S.A. 0196-9781/86 $3.00 + .00

Neuropeptide Y Receptors in Rat Brain: Autoradiographic Localization

J E A N - C L A U D E M A R T E L , * t S E R G E S T - P I E R R E t A N D R E M I Q U I R I O N .1

*Douglas Hospital Research Centre and Department of Psychiatry, McGill University 6875 LaSalle Blvd., Verdun, Quebec H4H IR3 Canada

tlnstitut National de la Recherche Scientifique en Santo and UniversitO du QuObec MontrOal, Quebec

R e c e i v e d 6 J a n u a r y 1986

MARTEL, J.-C., S. ST-PIERRE AND R. QUIRION. Neuropeptide Y receptors in rat brain: Autoradiographic localization. PEPTIDES 7(1) 55--60, 1986.--Neuropeptide Y (NPY) receptor binding sites have been characterized in rat brain using both membrane preparations and receptor autoradiography. Radiolabelled NPY binds with high affinity and specificity to an apparent single class of sites in rat brain membrane preparations. The ligand selectivity pattern reveals strong similarities between central and peripheral NPY receptors. NPY receptors are discretely distributed in rat brain with high densities found in the olfactory bulb, superficial layers of the cortex, ventral hippocampus, lateral septum, various thalamic nuclei and area postrema. The presence of high densities of NPY and NPY receptors in such areas suggests that NPY could serve important functions as a major neurotransmitter/neuromodulator in the central nervous system.

Neuropeptide Y Alzheimer's disease Catecholamines Receptor autoradiography

NEUROPEPTIDE Y (NPY) is a newly isolated member of a family of related peptides that include peptide YY (PYY) [52,53] and various pancreatic polypeptides (PP) [26, 29, 30]. NPY is one of the most highly concentrated and widely distributed peptides in the mammalian nervous system, including man [1, 4, 9, 14, 34, 40, 48, 50, 51]. This suggests that NPY could be a major neurotransmitter/neuromodulator in the brain [17,38]. Already, various pharmacological [3, 15, 16, 17, 18, 21, 25, 32, 33, 35, 38] and behavioral [2, 10, 28, 49, 57] effects elicited by NPY and/or related peptides support this hypothesis. For example, it is known that NPY produces a dramatic induction of feeding behavior [10, 28, 49], shifts circadian rhythms [2,57], alters the release of pituitary hor- mones [25,38] and modulates brain controls of cardiovascular parameters [16, 17, 18, 38]. The high concentration of NPY containing fibers in the cerebral cortex, hippocampus, septum and amygdala [9, 17, 38] also suggest possible roles for NPY in higher cognitive functions and in the mediation of emotional behavior [38]. Moreover, the co-localization of NPY and catecholamines in various noradrenergic and ad- renergic cell groups [17, 24, 38] suggests important functions for this peptide in autonomic regulation and in modulation of catecholaminergic pathways [17, 24, 38].

The multiple actions of NPY in the brain are probably initiated by binding to high affinity receptor sites. However, very little is currently known about these putative receptor binding sites [7, 47, 54, 55]. We report here for the first time on the binding characteristics of NPY and its fragments and on the receptor distribution of highly specific and selective NPY receptors in rat brain.

METHOD

Membrane binding assays were performed as follows: Male Sprague-Dawley rats (200-250 g) were killed by decapitation and the brain (minus cerebellum) was rapidly re- moved, placed on ice, homogenized (25:1, w/v) in Krebs/ Ringer buffer [41] pH 7.4 at 25°C, using a Brinkman Poly- tron (setting 6 for 15-20 sec) and centrifuged at 18,000 × g for 15 min. The supernatant was discarded and the precipitate rinsed, resuspended and centrifuged again. The final pellet was rinsed and resuspended in Krebs-Ringer buffer to give a protein concentration [31] of 12-18 mg/ml of membrane preparations. For binding assays, 100 #l of membrane preparation were incubated at 25°C for 120 min in a final volume of 500/zl containing Krebs buffer [41], pH 7.4 0.1% bovine serum albumin (Sigma), 0.05% bacitracin (Sigma) and various concentrations (0.05-20.0 nM for sat- uration experiments) or 0.5 nM [3H]NPY (ligand selectivity) (87 Ci/mmol; Amersham International Co.). Incubations were terminated by rapid filtration through Whatman GF/C filters (plus three 3 ml washes with cold buffer) previously soaked for at least 2 hours in 1.0% polyethyleneimine to reduce binding to filters. Specific binding was calculated as the difference in radioactivity bound in the presence and absence of 1.0 g.M NPY. Binding of the ligand to filters was quantitated by counting filters in 5 ml Scinti-Verse II (Fisher Scientific, Canada) scintillation cocktail. At 0.5 nM the amount of specifically bound ligand represent 70-75% of total binding (4518_ + 139 dpm in absence vs. 1161 -+35 dpm in presence of 1.0/zM NPY, n=3). NPY and NPY fragments

~Requests for reprints should be addressed to Dr. Rrmi Quirion, Douglas Hospital Research Centre, 6875 LaSalle Blvd., Verdun, Qurbec H4H 1R3 Canada.

55

56 MARTEL, ST-PIERRE AND QUIRION

TABLE 1 COMPARATIVE POTENCIES OF NPY, ITS FRAGMENTS, RELATED

AND UNRELATED PEPTIDES, AND DRUGS ON [3H]NPY BINDING TO RAT BRAIN MEMBRANE PREPARATIONS

Peptides IC~0 nM

[:IH]NPY Binding

Relative Relative Potency Potency Bioassay~

NPY 0.75 ± 0.09 100 NPY (2-36) 3.50 +_ 0.20 20 NPY (16-36) 95 ± 8.7 0.7 NPY (19-36) 10l ± 11.4 0.7 NPY (25-36) 2335 ± 255 0.03

PYY 3.05 ± 0.15 23 HPP* 620 ± 65 0.1 APP* > 10,000 <0.01

100 8.1 0.7 0.4

<0.1

IC50 is the concentration of competitor needed to displace 50% of specifically bound ligand. The following peptides and drugs were unable to displace [3H]NPY binding to rat brain membranes at concentrations up to 1.0 /xM: [Met~]-enkephalin, angiotensin II, substance P, substance K, somatostatin, clozapine, butaclamol, mus- cimol, cinanserine, prazosin, hemicholinium-3, amytriptyline, car- bachol, clonazepam, propranolol, nicotine, isoproterenol, haloperi- dol, clonidine, aspartic acid, nifedipine and verapamil. All results represent the mean --- SEM of three experiments, each in triplicate.

*HPP, human pancreatic polypeptide; APP, avian pancreatic polypeptide.

~Relative potency in the perfused heart bioassay as reported by Rioux et al. [46].

have been synthesized in our laboratories (Martel e t a l . , in preparation). Other peptides were obtained from the Institut Armand Frappier, Laval, Quebec, Canada. Rat brain sections were prepared for autoradiography as described before [22,42]. Briefly, male Sprague-Dawley rats (200-250 g) were killed by decapitation, and their brains rapidly immersed in 2-methyl-butane at -40°C, mounted on cryostat chucks and cut into 20/~m thick sections at -14°C. All sections were thaw-mounted on precleaned gelatin-coated slides and then stored at -80°C until used. For binding experiments, sections were preincubated for 60 min at 25°C in Krebs-Ringer buffer [41] at pH 7.4 and then incubated for 120 min at 25°C in the same buffer containing 0.1% bovine serum albumin, 0.05% bacitracin and either 0.1 nM [1251] Bolton-Hunter NPY (2000 Ci/mmol, Amersham International Co.) or 2.5 nM [3H]NPY. At the end of the incubation, slides were trans- ferred sequentially through 4 rinses (4 min in each) of cold incubation buffer and dipped in distilled water to remove ions. Incubated slides were then dried and tightly juxtaposed against tritium-sensitive film (Ultrofilm; LKB Instruments). After appropriate exposure, films were processed and analyzed as described before [42]. Sections were stained with cresyl violet to facilitate anatomical identification of brain structures [39]. Specifically bound [125I]NPY was eval- uated as the difference in binding seen in absence and presence of 1.0/xM NPY.

RESULTS AND DISCUSSION

In the first series of experiments, we characterized NPY

receptor binding sites in rat brain membrane preparations. Under optimal conditions (see the Method section for de- tails), [3H]NPY binds with high affinity and specificity to an apparent single class of saturable sites in brain membrane preparations. The apparent affinity (Kd) is in the low nanomolar range (1.8_+0.2 nM; Hill coefficient of 0.92) with a maximal capacity (Bm~x) of 335.5 -+ 32 fmol/mg protein (n = 3 ).

To characterize NPY binding pharmacologically, we examined the relative potencies of various NPY fragments, related and unrelated peptides, and drugs in inhibiting [3H]NPY specific binding to rat brain membrane preparations. As shown in Table 1, various NPY fragments exhibit different potencies on NPY binding sites. NPY., :~, possess only one fifth of the activity of NPY, demonstrating the criti- cal importance of the N-terminai tyrosine residue for the binding of NPY to its receptors. All other C-terminal fragments tested (Table 1) are weak competitors on [aH]NPY binding, again showing that the N-terminal portion of the molecule is essential to preserve the affinity of NPY for its receptor binding sites.

The structurally related peptide PYY is a fairly potent competi tor on pH]NPY binding sites while other related peptides such as HPP and APP are very much weaker (Table 1). Other unrelated peptides and drugs are unable to act on [3H]NPY binding sites, even at micromolar concentrations (Table 1).

Interestingly, the relative potency of NPY fragments in our binding assay correlates with their potencies in the guinea pig heart bioassay [46]. It has been shown that NPY > NPY~_:~, > NPYI6-:,~ > NPY~.~ :,~ > > NPY.,~ :~6 in increas- ing the pressure of perfusion in the guinea pig heart [461. This strongly suggests similar structural requirements for both central and peripheral NPY receptors. However, it has been shown that PYY is more active than NPY in certain tissues and that the pancreatic polypeptides have powerful effects in some bioassays [3, 17, 32, 38]. Thus, the existence of distinct classes of NPY, PYY and PP receptors cannot be excluded at the present time.

The autoradiographic distribution of ['z:q]BH-NPY binding sites is shown in Figs. 1 and 2. We also observed that [3H]NPY binding sites (not shown) are identically distributed as to [125I]BH-NPY sites, suggesting that both ligands bind to the same population of sites in the rat brain. As shown in Fig. 1, very high densities of NPY binding sites are present in the hippocampus, especially in the oriens layer and the stratum radiatum. The granular cell layer of the den- tate gyrus and the pyramidal cell layers of the hippocampus contain much lower densities of NPY binding sites (Fig. 1). At this level, moderate densities of sites are also found in the superficial layers (laminee I and II) of the cortex, portions of the lateral geniculate nucleus, ventral tegmental area and mammillary nuclei (Fig. 1).

Other areas that are highly enriched with [12al]BH-NPY binding sites include the anterior olfactory nucleus (Fig. 2A), lateral septum (Fig. 2B), superficial layers of various cortical areas (Fig. 2B, 2C), stria terminalis (Fig. 2C), various thalamic nuclei including the centro-medial, centro-lateral, antero-dorsal and paratenial nucleus (Fig. 2C), substantia nigra, pars compacta (Fig. IA) and pars lateralis (not shown) and area postrema (Fig. 2D). Moderate densities of NPY binding sites are seen in the frontal cortex (Fig. 2A), olfactory tubercule (not shown), pre-mammillary nuclei of the hypothalamus (not shown), medial geniculate nuclei (not shown), inferior colliculus (not shown), and inferior olive (Fig. 2D). Low to moderate densities of sites are found in the striatum (Fig. 2B,

NEUROPEPTIDE Y RECEPTORS 57

FIG. I. Photomicrographs of the distribution of [12~I]Bolton-Hunter Neuropeptide Y binding sites in rat brain at the level of the hippocampus. Adjacent sections were saturated in a buffer containing 0.1 nM [nSI]BH-NPY in the absence (A) and presence (B) of 1.0 tiM unlabelled NPY and then placed side by side in a cassette and exposed with a LKB Ultrofilm for 4 days. Abbreviations used: DH, dorsal hippocampus; MM, medial mammiilary nucleus; OR, oriens layer of the hippocampus; RD, stratum radiatum of the hippocampus; SNC, substantia nigra pars compacta; VH, ventral hippocampus; and VTA, ventral tegmental area.

2C), paraventricular nucleus of the hypothalamus (not shown), amygdala (not shown), and dorsal horn of the spinal cord (not shown). Low densities are observed in most re- maining hypothalamic nuclei (not shown), globus pallidus (Fig. 2B), superior colliculus (Fig. IA), central gray (Fig. 1A) and cerebellum (Fig. 2D). White matter areas such as the corpus callosum are devoid of specific binding sites (Fig. 2).

The respective distribution of NPY-like immunoreactivity [1, 4, 9, 14, 34, 40, 48, 50, 51] and NPY receptor binding sites shows interesting similarities and differences. For example, the very high densities of NPY and NPY receptors in the cerebral cortex and the hippocampus suggest roles for this peptide in higher cognitive functions [17,38]. Interestingly, it has been recently shown that cortical NPY-like immunoreactivity is not markedly altered [5,8] or decreased [6] in senile dementia of the Alzheimer type while it is increased in the substantia innominata [5]. NPY-like immunoreactivity, is also present in neuritic plaques in Alzheimer's disease [13]. Since it is believed that the cholinergic substantia innominata-cortical pathway is markedly affected in Alzhei- mer's disease [12, 36, 56], it could suggest that NPY may have an important role in the control of the activity of this pathway. Similarly, the high concentrations of NPY-like immunoreactivity and NPY receptors in the septum and hip-

pocampus could indicate that NPY modulates.the activity of the cholinergic septo-hippocampal pathway. Already, Col- mers et al. [11] have shown that NPY modulates excitatory transmission in the hippocampus by a presynaptic mechanism. Thus, possible interactions between NPY and specific cholinergic pathways should be investigated and further studies on NPY and NPY receptors in Alzheimer's disease are certainly warranted. The high densities of NPY and its receptors in the septum and the hippocampus also suggest roles for this peptide in the limbic system, although the possible effects of NPY on relevant behaviors remain to be established.

However, mismatches between the distribution of NPY and NPY receptors are apparent in various brain regions including the hypothalamus. For example, while very high densities of NPY-like fiber terminals are present in the suprachiasmatic nucleus (SCN) [9, 17, 38], only low to moderate densities of binding sites are observed in this region. This is somewhat surprising since direct injections of NPY into these areas have been shown to induce behavioral effects such as shifts of circadian rhythms [2]. This could be due to the existence of another class of receptors in this hypothalamic nucleus or to the presence of binding sites on adjacent brain structures [27]. Such apparent mismatches are not ex-

58 MARTEL, ST-PIERRE AND QUIRION

A

C

FR

tip

r

D

IO

FIG. 2. Photomicrographs of the distribution of [le:'l]Bolton-Hunter Neuropeptide Y binding sites in rat brain at the level of the forebrain (A), striatum (B and C) and brain stem (D). Sections were incubated in a buffer containing 0.1 nM ['2~I]BH-NPY. Following incubations, sections were placed side by side in a cassette and exposed with an LKB Ultrofilm for 4 days. Abbreviations used: AD, anterodorsal nucleus of the thalamus; AO, anterior olfactory nucleus; AP, area postrema; CE, cerebellum; CL, claustrum; CM. centro-medial nucleus of the thalamus; CP, caudate putamen (striatum); EP, external plexiform layer of the olfactory bulb; FR, frontal cortex; GP, globus pallidus; IO, inferior olive: LS, lateral septum; and ST, stria terminalis.

clusive to NPY and similar situations have been reported for various other neuropeptides [19, 20, 23] as well as classical neurotransmitters [23, 27, 43, 44, 45]. Also, NPY- immunoreactive fibers innervating the SCN have been shown to arise from perikarya located in the ventral lateral geniculate nucleus (VLGN) of the thalamus [37], which is enriched in NPY binding sites. It is possible that the most prominent effect on NPY on the SCN could be through the activation of receptors located in the VLGN resulting in a

stimulation of the VLGN-SCN pathway. Future studies should help to determine these points.

Finally, the high densities of NPY binding sites in the area postrema is of interest since NPY has been shown to induce various cardiovascular effects following central and peripheral injections [16, 18, 32, 33]. Moreover, the high incidence of co-localization between NPY and catecholamines [17, 24, 35, 38] suggests possible roles for NPY in autonomic regulation. We are currently investigating the effects of selective

N E U R O P E P T I D E Y R E C E P T O R S 59

les ions of ca t echo lamine rg i c p a t h w a y s on N P Y and N P Y recep to r s in r e l evan t b r a in regions . This could help to b e t t e r u n d e r s t a n d the func t iona l s ignif icance o f NPY- c a t e c h o l a m i n e in te rac t ions .

In s u m m a r y , high aff ini ty, pharmaco log ica l ly specif ic N P Y b ind ing si tes are p r e s e n t in d i sc re te a reas of the ra t bra in . The high dens i t i es o f si tes in cor tex , h i p p o c a m p u s , s ep tum, subs t an t i a n igra and var ious tha lamic nuclei sugges t tha t N P Y might be i nvo lved in the in tegra t ion o f h igher cog- n i t ive func t ions and in the med ia t i on of emot iona l behav io r s . Thus , s tudies on possible roles of N P Y and N P Y receptors

in the et iology of va r ious neuro logica l and psych ia t r i c dis- eases such as A l z h e i m e r ' s d i sease and s c h i z o p h r e n i a are cer- ta in ly war r an t ed .

ACKNOWLEDGEMENTS

This research project was supported by a grant to Rrmi Quirion from the Medical Research Council of Canada (MA-8580). Rrmi Quirion is a "Chercheur-Boursier" from "Le Fonds de la Recherche en Sant6 du Qurbec." The secretarial assistance of Mrs. D. Vetro and Mrs. J. Currie is acknowledged.

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neuropeptide y receptors in rat brain: autoradiographic localization

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