Neuroscience Letters, 75 (1987) 141 146 141 Elsevier Scientific Publishers Ireland Ltd.

NSL 04504

Distribution of neuropeptide Y receptors in the rat hippocampal region

Christer K6hler, Marianne Schultzberg* and Ann-Cathrin Rades/iter

Astra Alah AB, Research and Development Laboratories, Department o]" Neuropharmaeology, SOdertiille ( Sweden )

(Received 10 November 1986; Revised version received 8 December 1986; Accepted 10 December 1986)

Key words. Neuropeptide tyrosine; [~H]NPY; Receptor; Hippocampal region; Rat; Autoradiography

The distribution of binding sites for neuropeptide Y (NPY) was studied in the rat hippocampal region by using [3H]NPY together with quantitative in vitro receptor autoradiography. The highest density of specifically bound [3H]NPY was found in regio superior and regio inferior of Ammon's horn. Within these fields, stratum oriens, stratum pyramidale and stratum radiatum harboured the highest densities of [~H]NPY binding while stratum moleculare was relatively poor in [3H]NPY binding sites. In area dentata. the highest density of [3H]NPY binding was found in the inner one third of the molecular layer. In the presubiculum and in the entorhinal area, the outer two layers were slightly more enriched in [3H]NPY binding sites than were the deep layers. In all hippocampal subfields a clear gradient of increased [3H]NPY binding was found at successively more ventral levels.

Neuropeptide Y (NPY), a 36-amino acid peptide of the pancreatic polypeptide family [1 l, 12], is present in large quantitites in most cortical areas of several species [1, 2, 5, 7], including man [3, 4]. Immunohistochemical studies have localized NPY- immunoreactivity (NPY-i) to non-pyramidal neuronal somata and axonal processes throughout all cortical fields, including the hippocampal region [4, 5, 7]. The role(s) played by NPY in the neocortex and the hippocampus is unknown at present but its presence in hippocampal interneurons as well as in certain hippocampal afferents [8], suggest that NPY may participate in the modulation of neurotransmission along the intrinsic trisynaptic hippocampal circuit. Recently, binding sites for 125I-NPY were described and characterized in synaptic membranes of the rat cerebral cortex, hippocampus and hypothalamus [13] and, thus, there is evidence that receptors for NPY may exist in the rat brain. In the present study, we have analyzed the regional distribution of [3H]NPY-binding sites within the hippocampal region of the rat by using the method of quantitative in vitro receptor autoradiography.

Male Sprague-Dawley rats (Anticimex, Sollentuna, Sweden; weight 200 g) were

*Present address: Department of Pathology, Huddinge Hospital, Huddinge, Sweden. Correspondence: C. K6hler, Astra Alab AB, Research and Development Laboratories, Department of Neuropharmacology, S6dert/ilje, Sweden.

0304-3940/87:$ 03.50 © 1987 Elsevier Scientific Publishers Ireland Ltd.

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decapitated, their brains rapidly removed and frozen. Sections (20 l~m thick) were cut in a cryostat (Dittes, Heidelberg° F.R.G.) and collected onto chromalun-gelatine- coated glass slides. The sections were preincubated for 3 x 5 min in 20 mM HEPES buffer (pH=8.4) containing 1.8 mM CaCI2, 1.05 mM MgCI2, 1.37 mM NaC1, 2.68 mM KC1 and 1 g/1 glucose. After this preincubation the sections were transferred to the same buffer which contained 0.1 mM Bacitracine (Sigma, St. Louis, U.S.A.), 0.5% bovine serum albumin and different concentrations (1 or 2 nM) of [-~H]NPY ([3H]-N-propyonyl(-NPY) (Amersham; spec. act. 72.5 Ci.mmol--~) either alone or in the presence of 1 or 10/tM of the unlabelled peptide (CRB, Cambridge, England) to define specific binding. After incubation at room temperature for 30 min, the sec- tions were washed 5 x 2 min in HEPES at +4°C and thereafter dipped in distilled water for 20 s. The sections were dried in a stream of cold air, put in contact with tritium sensitive film (3H-Ultrofilm, LKB, Solna, Sweden) and exposed for 8 weeks at -20°C . The autoradiograms were analyzed by using an IBAS 2000 image ana- lyzer, equipped with a program which converted optical densities to fmol.mg - i tissue by using calibrated plastic tritium standards (Amersham) co-exposed with the tissue sections. In a separate set of experiments, tissue sections were incubated in different concentrations of [3H]NPY in the presence or absence of 10/LM unlabelled NPY. The sections were scraped off the slides and the radioactivity counted in a Packard scintillation counter.

The biochemical experiments showed that the binding of [3H]NPY to tissue sec- tions was saturable with a Kd ranging from 1.5 to 2 nM in two separate experiments. Addition of ! #M unlabelled NPY reduced the total binding by 30% indicating that a high degree of non-specific binding contributed to the total binding. The autoradio- grams revealed a heterogeneous pattern of [3H]NPY binding in the hippocampal re- gion. Approximately 50% of the total binding was non-specific as determined by ad- dition of 1/tM unlabelled NPY. Increasing the concentration of unlabetled NPY to 10/~M resulted in blockade of approximately 70% of the total [3H]NPY binding when measured autoradiographically within the hippocampal region. Compared to the other brain areas, the Ammon's horn and area dentata were found to be rich in speci- fically bound [3H]NPY. Within the Ammon's horn, regio superior and regio inferior contained the highest density of specifically bound [3H]NPY. Most of the binding in these subfields occurred in stratum oriens and stratum radiatum (Fig. 1, Table I), while the stratum pyramidale and the stratum lacunosum-moleculare were relatively poor in [3H]NPY-binding sites. Regio inferior contained a high density of [3H]NPY- binding sites and the general distribution of these sites was the same here as in regio superior. A dorsal to ventral gradient in the [3H]NPY-binding was particularly strik- ing in regio inferior. At mid-temporal and ventral levels, there was high density of binding in the stratum radiatum of regio inferior (Fig. 1, Table I). Most of the [3H]NPY binding sites in the area dentata occurred in the inner one third of the mole- cular layer, while moderate binding was found in the outer two thirds of this layer. The hilus of the area dentata was relatively poor in [3H]NPY-binding sites (Fig. 1~ Table I) and there was far less dense binding of [3H]NPY in the individual retrohip- pocampal structures compared to the Ammon's horn and area dentata. The same

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was true for the rest of the neocortex. The highest densities of [3H]NPY-binding sites in the retrohippocampal region were found in the outer two layers of the entorhinal area, and in the pre- and the parasubiculum (Fig. 1, Table I), while the binding was sparse in the subiculum. The deep layers of all retrohippocampal subfields contained little specific [3H]NPY-binding. The binding in all lamina of Ammon's horn and area dentata, but not in the entorhinal area, increased significantly at temporal levels (Fig. 1, Table I).

Previous studies have demonstrated the presence of binding sites for NPY using chloramine-T-iodinated NPY [13] tritiated and 1251-Bolton Hunter-labeled [9] NPY

A

RS p

S S o

S g

RI . s m

EA P R EA E

NI

s

Fig. 1. Dark field photomicrographs of saginal (A, B) and horizontal (C, D) sections of the rat hippocam- pus showing the distribution of [3H]NPY-binding sites, a, angular bundle; EA, entorhinal area: f, limbria: H, hilus of area dentata; PRE, presubiculum; PS, parasubiculum; RI, regio inferior: RS, regio superior:

S, subiculum; SM, s tratum moleculare; SO, stratum oriens: Sp, stratum pyramidale; Sin, stratum molecu- late. Arrows mark the borders between different subfields.

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in membranes of rat brain, suggesting the existence of central NPY-receptors . In the

present study, N P Y - b i n d i n g sites have been visualized in the h ippocampal region

using [3H]NPY as the l igand in a subsa tura t ing concent ra t ion of 1 nM. In good

agreement with the earlier biochemical studies [9, 13], the present au toradiographic

findings show a relatively high density of NPY receptor sites in the h ippocampal for-

ma t ion compared to the other cortical and subcortical brain regions. The general pat-

tern of b inding reported here is in good agreement with a recently reported study

by Martel et al. [9] using Bolton Hun te r 125I-NPY as the ligand. The presence of [3H] -

N P Y - b i n d i n g sites in the h ippocampus adds to a rapidly increasing list of putat ive

TABLE I

THE DISTRIBUTION OF BINDING SITES FOR [3H]NPY (1 nM) WITHIN THE DIFFERENT SUBFIELDS OF THE RAT HIPPOCAMPAL REGION AS MEASURED AT DORSAL AND VEN- TRAL LEVELS USING QUANTITATIVE RECEPTOR AUTORADIOGRAPHY USING A SUB- SATURATING CONCENTRATION OF LIGAND

All measurements are the mean + S.E.M. of 6 readings in two separate experiments. The values have not been corrected for quenching. Specific binding was determined in the presence of 1/tM unlabelled NPY.

[3H]NPY specifically bound (fmol.mg-- ~ tissue)

Anatomical region Dorsal levels Ventral levels

Subiculum Stratum pyramidale 6.9+__0.2 12.3-+ 2.3 Stratum moleculare 6.3-+0.5 11.4+ 2.3

Regio superior Stratum oriens 12.5 + 0.5 19.9___ 0.5 Stratum pyramidale 12.1 + 1.2 21.5+ 2.5 Stratum radiatum 15.4 + 1.7 24.9 + 5.7 Stratum moleculare 8.1 + 0.5 12.1 + 2.4

Regio inferior Stratum oriens 11.9+0.5 29.3__+ 5.5 Stratum pyramidale/lucidum 10.0 ___ 1.8 19.0 + 4.3 Stratum radiatum 12.8 + 2.5 44.4 + 12.2

Area dentata Molecular layer 10.6+0.5 14.4+ 1.9 Hilus 8.8___ 1.8 13.5+ 0.5

Presubiculum Layers I-II 7.8+0.5 11.1_I+ 2.0 Layers III-VI 5.6+0.5 12.04- 2.2

Medial Entorhinal area Layer 2 7.3_+0.5 7.0+ 0.5

3 8.2__+0.8 11.6-+ 0.5 4~6 8.9_+1.4 10.6+ 1.7

Angular bundle 3.5 8.0

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peptide receptors being discovered within this structure. The distribution of binding sites for [3H]NPY and several other peptides may partly overlap within the hippo- campus but in general the overall distribution pattern is quite distinct for each pep- tide receptor examined so far.

Immunohistochemical studies have shown a rich NPY innervation of the rat hip- pocampus [7]. The highest density of NPY-immunoreactive axons is found in the entorhinal area, the pre- and the parasubiculum [7]. These areas contain, however, only low to moderate densities of [3H]NPY-binding sites. Furthermore, stratum oriens and stratum radiatum of the Ammon's horn, two lamina which are relatively poor in NPY-immunoreactive axons, have the largest number of [3H]NPY binding sites in the entire hippocampal region. The molecular layer on the other hand, which is conspicuously poor in [3H]NPY binding sites, harbours a large number of NPY- immunoreactive fibers [7]. This poor concordance between receptor distribution and innervation density for a neurotransmitter candidate, is a well known phenomenon described for several other neurotransmitter receptors, but the reason(s) for this phe- nomenon is not fully known at present.

The cellular localization of the [3H]NPY-binding sites in the hippocampus is not known. In regio superior and regio inferior, the highest density of binding occurs in stratum oriens and stratum radiatum, which are the dendritic zones of the basal and apical dendrites of the pyramidal cells, respectively. It is thus possible that NPY receptors are situated on these dendrites. However, a localization on commissural and intrinsic axons which terminate in these layers must be considered. A similar problem is encountered in the area dentata where a band of dense [3H]NPY binding is present in the inner molecular layer. This zone is occupied by numerous afferents of extrinsic as well as intrinsic origins and thus, experimental methods are required to disclose the anatomical locus of NPY receptors in this layer. In the retrohippo- campal region, [3H]NPY binding is found primarily in the outer two layers of the presubiculum and the entorhinal area. These layers give rise to the two most promi- nent association pathways in this part of the cortex. One of these, the perforant path, originates in the second layer of the entorhinal area [10] and exerts a powerful excita- tory drive upon the area dentata. The other is a projection from the presubiculum to those layers (layers 2, 3) of the EA which give rise to the perforant path [6]. The presence of NPY receptors in these, and in the other hippocampal layers just dis- cussed, may imply a role for NPY in the modulation of hippocampal neurotransmis- sion.

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