Quantitative receptor autoradiography demonstrates a differential distribution of neuropeptide-Y Y1 and Y2 receptor subtypes in human and rat brain

27

Brain Research, 631 (1993) 27-38 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00

BRES 19510

Quantitative receptor autoradiography demonstrates a differential distribution of neuropeptide-Y Y1 and Y2 receptor subtypes in human and rat brain P.S. W i d d o w s o n

*

Departments of Psychiatry and Neurosciences, Case Western Reseree UniL,ersity and MetroHealth Medical Center, Cle~,eland, OH 44109, USA (Accepted 20 July 1993)

Key words: Neuropeptide Y; Autoradiography; Yl-type receptor; Y2-type receptor; Human brain; Rat brain

Quantitative receptor autoradiography was performed on sections of rat and human brain using [tZSl]peptide YY ([1251]PYY) to measure the anatomical distribution of neuropeptide Y (NPY) receptors. Masking 571- and Y2-NPY subtypes with the agonists [Leu31,Pro34]NpY and NPY13_36, respectively demonstrated a differential distribution of Y1 and Y2 receptors between human and rat brain. In human brain, the highest density of [12SI]pYY binding was found in pyramidal layers (CA4-CA1) of hippocampus, head and tail regions of caudate nucleus, locus coeruleus and substantia nigra. There was moderate [lzSI]peptide YY binding to NPY receptors in the molecular layers of the hippocampus, frontal and temporal cerebral cortex, especially in superficial layers, anterior amygdala, central grey and inferior colliculus. Low levels of binding were observed in white matter. The selective YI receptor agonist, [Leu31,Pro34]NPY did not effectively reduce [125I]PYY binding to any human brain region examined except for approximately 20-40% of the binding sites in the molecular layer of the dentate gyrus, layer IV of the frontal cortex and the radiatum and oriens layers of the hippocampal complex. In contrast, the Ye agonist, NPY13_36 was effective at reducing [ 12SI]pYY binding in all human brain regions examined. In rat brain, high densities of [125I]PYY binding was measured in cerebral cortex, thalamus and inferior colliculus which was sensitive to [Leu31,Pro34]NPY. In contrast, high densities of the NPY13_36 sensitive binding was found in the hippocampus, striatum and nucleus accumbens. Medium to low densities of NPYI.~_3~ sensitive binding was found in medulla and pons. This data suggests that human brain contains primarily Y2-type NPY receptors with only a few regions expressing Yt-type receptors. No human brain region examined contained solely Yl-type receptors. In contrast to human brain, rat brain contains regions which express only Y1 receptors as well as regions containing only Y2 receptors and regions containing both Y1 and Y2 receptors.

INTRODUCTION It is now clear that the 36 amino acid neuropeptide Y (NPY) 23 produces its effects through the interaction at multiple NPY receptors 13'17'21. Originally the proposal for multiple NPY receptors was based on the ability of the C-terminal NPY fragment, NPY13_36, to pre-junctionally inhibit nerve stimulation-evoked contractions of rat vas deferens 25 and inhibit transmitter release 12 which was slightly less potent than its native NPY. In contrast, the post-synaptic actions of NPY, to directly stimulate the contraction of vascular smooth muscle, was not mimicked by the C-terminal NPY13_36 fragment 25. Hence it was suggested that the pre-syn-

aptic NPY receptors, which are sensitive to NPYI3_36, be classified as Yz-type receptors and the post-synaptic NPY receptors which are insensitive to NPYt3_36 as Y~-type NPY receptors 25. However, later studies demonstrated that Y2-type receptors are not selectively located pre-synaptically but also can exist at post-synaptic sites on smooth muscle 13,17,21 Binding studies using e i t h e r [125I]NPY or [125I]peptide YY (PYY) also provided tentative evidence for NPY receptor subtypes in rat brain 1'14'16'26. Using binding techniques and NPY~3_36 to discriminate YI receptors from Yz-type receptors, cell lines were discovered which express either Y~ or Yz receptors 22 Using these cell lines, a C-terminally modified NPY

* Corresponding author. Present address: Central Toxicology Laboratory, Zeneca Ltd., Alderley Park, Macclesfield, Cheshire SK10 4TJ, UK. Fax: (44) (625) 58-2897.

28 agonist, [Leu3~,Pro34]NpY, was found to have more than 100-fold more selectivity at the Y~-type receptors as compared to Y2 recep torS"6- Although binding studies show that NPY13_36 is only marginally selective for Y2-type receptors 5"6, it remains the only compound to date which has a greater affinity at Y2 receptors as compared to Yt receptors. Using [J25I]PYY or [~25I]NPY, autoradiographic studies were performed to anatomically localize the distribution of Y~ and Y2 subtypes in rat brain ~'2"3. In these studies, NPYI3_36 was used to mask Y2 receptors and [Leu31,Pro34]NpY was used to mask Yl-type receptors 1'2'3. [125I]PYY or [~25I]NPY binding to sections of rat brain was sensitive to low concentrations of [Leu31,Pro34]NpY in the cerebral cortex, but not in the hippocampus suggesting that the cerebral cortex contains Y~-type receptors whilst the hippocampus contains Yrtype receptors L2'3. The differential localization of NPY subtypes in rat brain was further substantiated with the report that [ms I]PYY or [~25I]NPY binding to rat hippocampus, but not cerebral cortex was sensitive to low concentrations of NPy~2'36. Thus rat brain contains NPY subtypes in different proportions in different brain regions ~'2'3'~6. Recently two peptide receptors have been cloned from rat tissue and fetal human brain with characteristics of Y~-type NPY receptors 7mu~ suggesting that Y~ receptors, like rat brain, can be localized to human brain. An earlier study comparing [3H]NPY binding to homogenates of rat and human frontal cortex showed similar characteristics in the pharmacology of NPY receptors between the two species 27. This study examines the relative distribution of [~25I]PYY binding to NPY receptors using quantitative receptor autoradiography in human and rat brain regions. [125I]PYY has chosen in preference to [~25I]NPY because [125I]PYY produces lower levels of non-specific binding ~4'~6, however NPY and PYY have demonstrated to have equal affinities at NPY receptors in both rat and human brain 27. In addition, [Leu3t,Pro34]NPY and NPYI3 36 were used to mask Y~ and Y2-type NPY receptors, respectively, to investigate and compare the regional distribution of NPY receptor subtypes in rat and human brain regions.

MATERIALS

AND METHODS

Anima& Male Sprague-Dawley rats (200-250 g) were housed in groups of four and maintained on a dark/light cycle with food and water available ad libitum under strict accordance to the NIH Guide for the Care and Use of Laboratory Animals. The rats were killed by decapitation immediately following carbon dioxide anesthesia according to approved procedures of the Animal Care Committee of Case Western Reserve University. The brains removed and placed in ice-cold

saline (0.9%) solution. The brains were frozen in isopentane ( - 50°C) which was cooled on dry-ice, and the brains stored at - 80°C. Twenty micron sections were cut from the rat brains at - 2 0 ° C and the sections mounted on gelatin-coated slides. The sections were dried overnight at 4°C and then stored at - 8 0 ° C until assayed.

Human brain Blocks of h u m a n brain (approximately 3 cm 3) containing various brain regions of interest, were dissected at autopsy from six subjects who died from myocardial infarctions (5 males, 1 female, age range 43-62 years, m e a n age = 56.5). Post-mortem intervals of the subjects before tissue pieces were frozen ranged from 4 to 25 h (mean = 14.8 h). NPY receptors in post-mortem h u m a n brain appear to be stable since there is no correlation between the density of NPY receptors against the post-mortem time interval to freezing tissue, up to a maximum of 35 h (data not shown). Toxicological screening to detect antidepressants, antipsychotics, barbiturates, benzodiazepines, opiates, stimulants, cannabinoids and alcohol were performed at autopsy. Five subjects did not have any drugs in their blood at autopsy, whilst one of the males tested positive for ethanol. The blocks of

TABLE I

Specific binding of 100 pM [1251]PYY to sections of human brain in the presence of either [Leu~I, Pro~4]NpY or NPY13_36 Results expressed as fmol b o u n d / m g protein. Mean_+ S.E.M. for 6 brains except where indicated. Experiments were performed in duplicate.

Brain region

Specific bound

Specific bound + Y1 agonist

Specific bound + Y2 agonist

Frontal cortex (BA 10) layers I - I I l layer IV layers V - V I Underlying white matter

8.8+_1.1 4.9 _+0.9 3.1_+0.3 1.7 _+0.2

7.3_+2.1 3.2 _+2.0 2.5_+0.3 1.3 + 0.8

2.6_+0.1 1.7 _+0,7 0.5_+0,2 0.3 + 0.6

15.5_+2.3 12.2-+3.4 6.1_+0.5 14.4 + 1.3 12.2_+4.5 1.8_+0.3 20.5 _+0.8 22.8_+2.4 3.6_+0.6

14.0_+1.8 11.1-+2.6 5.6_+0.8 13.9 _+ 1.0 10.7_+5.7 1.4_+0.6 17.9 _+1.7 18.9_+3.7 3.2_+0.8

4.6-+1.2 3.6+_2.2 1.5_+1.0 2.2 _+0.4 4.5_+ 1.3 0.3_+0.3 5.7 _+0.7 4.0_+0.9 0.8_+ 1.0

27.0_+2.7 28.1 _+2.4 31.4 _+2.9 21.5 _+ 1.3 11.1 +- 1.3 8.6 +_ 1.9 11.9 _+0.8 15.7_+ 1.2 4.7-+1.5 15.0+_2.1 7.4+_2.7 12.6+_3.5 6.4+_2.1 15.3-+3.5 2.1-+0.6 1.9_+0.4

25.9_+2.6 27.5 _+3.8 26.8 _+3.3 20.1 _+0.9 7.7 _+0.6 5.7 _+ 1.2 9.9 _+1.5 12.2_+ 1.7 4.3_+2.6 13.6_+2.6 7.7_+ 1.7 11.8_+2.9 7.0_+ 1.6 13.8_+3.0 1.8_+0.3 1.7_+0.4

4.4_+ 1.6 4.0 _+ 1.9 3.5 _+1.7 4.0_+ 1.6 3.5 _+0.7 1.5 _+0.4 3.7 _+0.6 4.4_+0.9 2.0_+1.1 3.7_+ 1.4 3.6_+ 1.0 4.7_+2.1 2.2_+0.3 5.4_+2.4 1.0_+0.2 0.9_+0.3

Piriform cortex layers 1-3 layer 4 layers 5 - 6 Head of caudate nucleus Tail of caudate nucleus * Internal capsule Lateral amygdala Basolateral amygdaloid N. Surrounding white matter

Hippocampus Pyramidal CA4 layer Pyramidal CA3 layer Pyramidal CA2 layer Pyramidal CA1 layer Radiatum layer Oriens layer Dentate molecular layer Dentate granular layer Inferior colliculus * Substatia nigra * Periaqueductal grey * Medial raphe nucleus * Dorsal raphe nucleus * Locus coeruleus Reticular formation Medial longitudinal fasiculus

* Data obtained from only 2 subjects.

29 brain tissue were immediately frozen in isopentane cooled on dry-ice and stored at -80°C. Twenty p.m sections were cut from the brain tissue at -16°C and the sections thaw mounted on gelatin coated slides• Sections were dried overnight as with rat brain sections and stored at - 80°C.

Autoradiography The sections were briefly warmed to room temperature. Eight adjacent slides through each brain region were randomized and assigned to either total binding, binding with Y~ receptor masking, binding with Y2 receptor masking or non-specific binding. The sections were pre-incubated in modified Krebs-Ringer binding buffer (NaCI 137 mM, KCI 5.4 mM, KH2PO 4 0.44 mM, CaCI 2 1.26 raM, MgSO4 0.81 mM, HEPES 20 mM, bovine serum albumin 0.1%; pH 7.4) for 30 min at 20°C to remove any remaining endogenous ligand. The sections were then incubated with 100 pM [lZ5I]peptide YY

=. ~t 7"

(2200 Ci/mmol, NEN, DE, USA) in binding buffer containing bacitracin 0.05% (Sigma Chemical Co., St. Louis, MO), soybean trypsin inhibitor 0.05% (Sigma Chemical Co., St. Louis, MO) and dithiothreitol (DTT) 1 mM for 90 min at 20°C. Non-specific binding was defined with 1 /zM porcine NPY (Bachem Inc., Torrence, CA). Yt-Type NPY receptors were masked using 100 nM [Leu31,Pro34]NpY (Bachem Inc.) and Y2-type receptors were masked with 300 nM porcine NPYI3_36 (Bachem Inc.). Masking concentrations of [Leu3~,Pro34]NPY and NPY13_36 were chosen from the work of Aicher et al). At these concentrations, 100 pM [Leu3~,Pro34]NpY will fully displace [~25I]PYY from Y~-type receptors and NPYI3_36 will approximately displace 80% of binding to Ye-type receptors whilst displacing only about 20% of binding to Y:type receptors ~. The sections were washed twice in ice-cold binding buffer for 5 min each and the sections briefly dipped in ice-cold deionized water. The sections were rapidly dried under a stream of warm air and then juxtaposed against tritium-sensitive film (Ultrafilm, LKB) in X-ray

A

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Human Frontal Cortex

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Human Brain

Rat l~¥ontal Cortex

GP Amyg Rat Brain

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IC SN PAG LC Rat Brain

Fig. 1. [1251]PYY binding remaining in the presence of 100 pM [Leu31,Pro34]NpY (open columns) or 300 pM NPY13_36 (hatched columns). Data represented as percentage binding remaining with NPY agonists with respect to specific binding. (A) Binding in human and rat frontal cortex through layers I to VI. (B) Binding in rat and human hippocampus. CA4-3 = pyramidal cell layers CA4-3, CA2-1 = pyramidal cell layers CA2-1, Dm = molecular layer of dentate gyrus, Dg = granular layer of dentate gyrus, D = molecular and granular layer of rat dentate gyrus. (C) Binding in rat and human amygdala and basal ganglia. C-head = head of caudate nucleus, C-tail = tail of caudate nucleus, Amyg = amygdala, St = striatum, GP = globus pallidus. (D) Binding in rat and human midbrain and medulla oblongata regions. IC = inferior colliculus, SN = substantia nigra, PAG = periaqueductal grey region, LC = locus coeruleus.

30 cassettes for 20 h (human sections) or 4 days (rat sections) with autoradiography standards (American RadioChemical Company (ARC), St. Louis, MO). The autoradiograms were developed and fixed with Kodak GBX. The autoradiograms were analyzed using computer densitometry (Micro Computer Imaging Device (MCID), Imaging Technology, Brock University, St. Catherine, Ont., Canada). Each section was analyzed separately and the specific binding and binding remaining after agonist masking of NPY subtypes calculated from the density of binding to each of the sections. The amount of binding to sections in the presence of 1 p~M NPY (non-specific binding) were subtracted from the values of total binding. Binding to the NPY receptor subtypes were estimated from binding in the presence of either [Leu31,Pro34]NpY (binding to Y2 receptors) or NPYt3_36 (binding to Yt receptors) minus the non-specific binding. Sections were stained with Cresyl violet and the brain regions confirmed using the rat stereotaxic atlas js and h u m a n neuroanatomy manual 19.

RESULTS In human brain, there was a moderate amount of specific binding to sections of cerebral cortex obtained from the frontal lobes (BA 10) (Table I). The density of

A

[~25I]PYY binding was laminar dependent throughout the cerebral cortex. Specific binding was highest in the superficial layer I and then decreased throughout the deeper layers. There was a low, but detectable level of specific binding in the white matter directly underlying the cerebral cortex (Table I). The addition of [Leu31,Pro34]NpY to the buffer did not alter the specific binding of [~25I]pYY to frontal cortex except in layer IV where there was a 40% reduction in binding. In contrast, 300 nM NPYI3 36 was able to displace approximately 80% of specific binding throughout cortical layers except for layer IV where NPY~3_36 was able to displace only 65% of binding (Fig. 1, Table I). The highest density of [~2SI]PYY binding was observed in human hippocampus, as compared to all the other brain areas examined (Fig. 2, Table I). The regions of hippocampus with the highest NPY receptor densities were the pyramidal layers. Layers CA4, CA3 and CA2 contained approximately 20% more sites than

C

4

E

Fig. 2. Binding of [125I]pYY to sections through h u m a n hippocampus. V = lateral ventricle, C = tail of caudate nucleus, C A I - C A 3 = pyramidal layers of hippocampus, mol = molecular layer of the dentate gyrus. A = total binding, B = non-specific binding in the presence of 1 ~ M NPY, C = binding in the presence of 100 pM [Leu3t,Pro34]NpY, D = binding in the presence of 300 pM NPY~3 36-

31 the CA1 region (Fig. 2, Table I). The surrounding radiatum and oriens layers also contained high densities of NPY receptors, but these densities were approximately three-fold less than in pyramidal cell layers (Table I). There was a moderate amount of [125I]PYY binding in dentate gyrus with the granular layer showing approximately 50% higher binding as compared to the molecular layer (Fig. 2). As with the cerebral cortex, [Leu31,Pro34]NpY did not effectively reduce [125I]PYY binding to pyramidal layers, whereas the addition of NPY~3_36 reduced binding by approximately 70-80% (Fig. 1, Table I). In contrast to the pyramidal layers, [Leu31,Pro34]NpY was able to reduce the [~25I]PYY binding to radiatum and oriens layers and to the granular and molecular layers of the dentate gyrus by between 34-17% (Table I). NPY13_36 was less effective in competing for [125I]PYY binding in radiatum, oriens, dentate molecular and dentate granular layers as compared to [125I]PYY binding to pyramidal layers (Fig. 1, Table I).

ii!

There was a high level of binding to human amygdala (Fig. 3, Table I) with much lower binding to the surrounding white matter. Specific [1esI]PYY binding was approximately equal in the lateral amygdala area and basolateral nucleus (Fig. 3). Specific binding to surrounding piriform cortex was also high, being approximately 50% higher than that observed in frontal cortical layers (Fig. 3, Table I). Moderate amounts of [125I]PYY binding were observed in caudate nucleus, with approximately equal binding in the head (Fig. 4) and tail (Fig. 2) regions (Table I). Specific binding to the internal capsule was very low and was equal to levels measured in other regions containing white matter (Table I). NPYt3_36 reduced [t25I]PYY binding by 70-80% in all amygdaloid areas, caudate nucleus, piriform cortex and surrounding white matter whereas [Leu31,Pro34]NpY was ineffective at substantially [125I]PYY reducing binding to any of these brain regions (Figs. 1 and 3, Table I).

I!!U ¸

Fig. 3. Binding of [125I]pYY to sections through h u m a n amygdala and piriform cortex. Pi = piriform cortex, BL = basolateral amygdaloid nucleus, L = lateral amygdaloid nucleus. A = total binding, B = non-specific binding in the presence of 1 /zM NPY, C = binding in the presence of 100 pM [Leu31,Pro34]NPY, D = binding in the presence of 300 pM NPYI3_36.

32

C

Fig. 4. Binding of [1251]PYY to sections through h u m a n caudate nucleus. C = head of caudate nucleus, ic = internal capsule. A = total binding, B = non-specific binding in the presence of 1 /zM NPY, C = binding in the presence of 100 pM [Leu31,Pro34]NpY, D = binding in the presence of 300 pM NPY13_36.

In human medulla oblongata, the highest amount of binding was observed in the ventral portion of the substantia nigra (Fig. 5) and locus coeruleus (Fig. 6) with moderate binding to the inferior colliculus, central grey, medial and dorsal raphe nuclei (Fig. 5). [125I]PYY binding throughout the reticular formation was moderate with numerous streaks of low binding in white matter which constitutes the fibre tracts (Fig. 6). Binding to the medial longitudinal fasciculus and superior cerebellar peduncle both of which are large fibre tracts was the lowest measured in the medulla oblongata (Table I, Fig. 5). NPY13_36 was effective at reducing the specific binding in all medullary regions examined whereas [Leu31,Pro34]NpY was virtually ineffective (Fig. 1, Table I). The distribution of [125I]pYY binding to rat brain regions were generally similar with a high density of binding in hippocampus, locus coeruleus, inferior colliculus and cerebral cortex, but low levels of binding in white matter (Figs. 7-9). Specific binding was approxi-

mately equal between rat and human frontal cortex and between the rat and human inferior colliculus. [~25I]pYY binding to rat cerebral cortex, like the binding to human cerebral cortex, varied throughout the layers with the highest binding being found in layers I and then decreasing through to layer VI. Binding to rat hippocampus, striatum, amygdala, substantia nigra, periaqueductal grey and locus coeruleus was observed to be much lower than their comparative human brain regions (Table II). In contrast to human brain regions, [Leu31,Pro34]NpY was effective in reducing [IE5I]pYY binding in a number of rat brain regions (Figs. 1, 7-9, Table II). Regions of rat brain which had a large reductions in [125I]PYY binding with [Leu31,Pro34]NPY (80-90%), but only a small reduction NPYI3_36 (1520%) include cerebral cortex, nucleus accumbens and inferior colliculus (Fig. 1). Specific binding to rat thalamic regions, hypothalamic regions, striatum, dentate gyrus of the hippocampus, superior colliculus, periaqueductal grey, olfactory

33

C

.~

i!ii!~II

Fig. 5. Binding of [125I]PYY to sections through the human medulla oblongata. Aq = aqueduct, CG = central grey, D R = dorsal raphe, MR = medial raphe, mlf = medial longitudianl fasicutus, C = inferior colliculus, SNL = substantia nigra lateral part, scp = superior cerebelar peduncle. A = total binding, B = non-specific binding in the presence of 1 p.M NPY, C = binding in the presence of 100 pM [Leu31,Pro34]NPY,

D = binding in the presenceof 300 pM NPY13_36.

tubercle and ventral tegmental area were reduced with both [Leual,Proa4]NPY and NPY13_36 (Fig. 1, Table II). The reduction in specific binding with NPYI3_36 was slightly greater than with [Leu3~,Pro34]NpY in rat hypothalamus and ventral tegmental area (70-90%) whilst binding to rat thalamus and superior colliculus was reduced to a greater extent with [Leu31,Pro34]NpY as compared to NPY13_36. Finally rat brain regions where NPY13_36 produced large reductions in specific binding, whilst [Leu31,Pro34]NpY was largely ineffective, include hippocampal pyramidal layers CA1-4, substantia nigra and amygdala (Fig. 1, Table II). DISCUSSION To our knowledge, this is the first paper which attempts to quantitate subtypes of NPY receptors in human brain. The demonstration of high densities of NPY receptors in human brain regions such as sub-

stantia nigra, locus coeruleus, caudate nucleus, hippocampus and amygdala suggests that NPY may be involved in a variety of functions in human brain. In many brain regions, the density of binding to NPY receptors was higher in human brain areas as compared to rat brain areas. The distribution of [125I]pYY sensitive NPY receptors in rat brain was generally the same as reported previously 1'2'1s'16 with the highest density of binding in hippocampus and the lowest in white matter. In rat brain, there is a relatively low density of NPY receptors in striatum but a moderate level of binding in substantia nigra, as compared to binding to the same regions in human brain. This suggests that the role of NPY in the nigrostriatal system may be more important in human brain as compared to rat brain. There was a high density of NPY receptors in the locus coeruleus of the medulla oblongata, which is the source of the majority of ascending noradrenergic neu-

34 ronal projections in mammalian brain. A moderate amount of NPY receptor density was also observed in both median and dorsal raphe nuclei, which constitutes the ascending serotonergic neuronal projections in mammalian brain. This suggests an important role of NPY systems in noradrenergic and serotonergic regulation in human brain at the level of the cell body. There was also a high density of NPY receptors in the rat locus coeruleus which suggests a similar role in the regulation of noradrenergic activity in rat and human brain. Intracerebral administration of NPY to rats has been demonstrated to alter the turnover of both serotonin and norepinephrine in forebrain terminal fields of the monoamine projections 24. Since there is a moderate to high density of NPY receptors in regions containing the monoamine cell bodies, the NPY-induced reductions in monoamine turnover may be caused by a direct effect of NPY on the cell bodies of the monoamine neurons through a reduction in cell firing.

The highest density of NPY receptors in both rat and human brain was observed in pyramidal layers of the hippocampus although the binding of [125I]PYY was approximately double in the human hippocampus as compared to the rat. Such a high density of NPY receptors in rat and human hippocampus suggests that NPY is may be an important neuromodulator in the process of memory formation in humans, as has been demonstrated in rats 4. [Leu31,Pro34]NPY was ineffective in reducing [~25I]PYY binding to a majority of human brain regions except in molecular and granular layers of the dentate gyrus and radiatum and oriens layers of the hippocampus were [Leu31,Pro34]NpY produced more than 20% reductions in binding. In addition, [Leu31,Pro34]NpY reduced the [125I]PYY binding to layer IV of the frontal cerebral cortex by approximately 40%. There are two possibilities for the failure to detect large reductions in specific binding with [Leu31,Pro34]NpY in more brain regions. Either the human brain contains a predomi-

C

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i

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Fig. 6. Binding of [125I]PYYto sections of medulla oblongata. IV = fourth ventricle, LC = locus coeruleus, mlf = medial longitudinal fasciculus, Rt = reticular area. A = total binding, B = non-specific binding in the presence of 1 p.M NPY, C = binding in the presence of 100 pM [Leu31,Pro34]NpY, D = binding in the presence of 300 pM NPY13_36.

35 nance of Y2 type NPY receptors or human brain Y~ type receptors may be less sensitive to [Leu31,Pro 34] NPY as compared to rat Y~ receptors. However, the recent cloning of a Yl-type NPY receptor from human fetal tissuesTM and examination of its pharmacological profile shows that the human Y~ receptor has a high affinity for [Leu31,Pro34]NpY, as does the rat Y1 homology ~°. Thus the Y1 receptor must have a limited distribution in adult human brain. A number of human brain regions, such as the inferior colliculus, locus coeruleus and substantia nigra which contain predominantly the Y2 subtype, as characterized by low [Leu31,Pro34]NpY sensitivity, also retain significant amounts of binding in the presence of NPY13_36. It is probable that [tzSI]PYY binding could be further reduced if the concentration of NPYt3_36 had been increased above 300 nM. However, competition studies have shown that above 300 nM, the selectivity of NPY13_36 is greatly reduced resulting in the displacement of PYY and NPY from Y~ receptors. The reason why NPYI3_36 failed to reduce [t25I]PYY bind-

13

ing in the locus coeruleus to the same degree as in the pyramidal layers of the hippocampus is unknown, but may suggest the presence of a third NPY receptor subtype in the locus coeruleus that is labelled by [125I]pYY and is less sensitive to both NPY13_36 and [Leu31,Pro34]NPY. There is now emerging evidence that the family of receptors for NPY and PYY may be greater than the well characterized Y1 and Y2-types. Binding studies in various tumor cell lines has shown that the affinity of NPY, PYY and NPY analogues cannot be explained assuming only Y1 and Yz-type receptors s. In addition, a peptide receptor has been cloned from rat locus coeruleus with a high affinity for NPY but a much lower affinity for PYY and has been classified as a Y3-subtype z°. In rat brain there are regions which contain either Y1 or Y2 sites, with a number of brain regions containing both subtypes. The autoradiographic neuroanatomical distribution of Y1 and Y2 receptors in rat brain described in this study closely matches the distribution described previously ~'2'3. One of the major differences

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Fig. 7. Regional binding of [t25I]PYY to sections of rat brain at 1.2 mm anterior to the bregma (Paxinos and Watson, 1982). C = cerebral cortex, cc = corpus callosum, St = striatum. A = total binding, B = non-specific binding in the presence of 1 /xM NPY, C = binding in the presence of 100 pM [Leu31,Pro34]NPY, D = binding in the presence of 300 pM NPY13_36.

36

C

Fig. 8. Regional binding of [lZSl]pYY to sections of rat brain at - 5 . 8 m m anterior to the bregma (Paxinos and Watson, 1982). PC = parietal cortex, Hp = hippocamous, Sn = substantia nigra. A = total binding, B = non-specific binding in the presence of 1 txM NPY, C = binding in the presence of 100 pM [Leu31,Pro34]NPY, D = binding in the presence of 300 pM NPYI3_36.

that exists between rat and human brain is the NPY subtype that is found in cerebral cortex. In rat cerebral cortex, [t25I]PYY specific binding is almost completely abolished by [Leu31,Pro34]NpY whereas binding to human cortex is relatively unaltered. In contrast NPYI3_36 produces a small decrease in binding in rat cerebral cortex but a large reduction in binding in human cerebral cortex. This suggests that the rat cerebral cortex contains primarily Y1 type receptors whilst the human cortex contains the Yz subtype. A earlier study which examined the binding characteristics of [3H]NPY in rat and human frontal cortex failed to observe a difference between the two tissues 27. However, since no compounds which are even marginally Y1 or Y2 selective were examined, the similarity in [3H]NPY binding between rat and human cortex is not surprising. [125I]PYY specific binding to both human and rat amygdala and pyramidal layers of the hippocampus are

sensitive to NPYt3_36 but not to [Leu31,Pro34]NpY suggesting that in both species, the predominant NPY subtype is the Y2 type. In rat brain, there is more than a 20% reduction in specific binding with both [Leu3t,Pro34]NpY and NPY13_36 in striatum, thalamus, hypothalamus, periaqueductal grey, superior colliculus and ventral tegmental area. This suggests that in these brain structures, there is a coexistence of both Y1 and Yz subtypes. Evidence is now emerging that intracerebroventricular administration of NPY which alters both body temperature and feeding behavior is mediated by multiple NPY receptors 9. Thus a number of behavioral changes attributed to NPY may be subclassified depending on the subtype of NPY receptor involved. In conclusion, we have demonstrated that rat and human brain differ considerably, regarding the distribution of NPY receptors in selective brain regions. The human brain contains primarily Y2-type receptors with

37

A

Fig. 9. Regional binding of [125I]pYY to sections of rat brain at - 8 . 8 mm anterior to the bregma (Paxinos and Watson, 1982). Ic = inferior colliculus, Lc = locus coeruleus. A = total binding, B = non-specific binding in the presence of 1 p.M NPY, C = binding in the presence of 100 pM [Leu31,Proa4]NpY, D = binding in the presence of 300 pM NPY13_36.

few regions containing Y1 receptors whilst rat brain, as a whole, contains more proportionate amounts of Y1 and Y2 receptors. Acknowledgements. This work was supported by a grant from the National Alliance for Research into Schizophrenia and Depression (NARSAD). The author wishes to thank Drs. G.A. Ordway and C.A. Stockmeier (Case Western Reserve University) for supplying the human brain tissue. REFERENCES 1 Aicher, S.A., Springston, M., Berger, S.B., Reis, D.J. and Wahlestedt, C., Receptor-selective analogs demonstrate NPY: PYY receptor heterogeneity in rat brain, Neurosci. Lett., 130 (1991) 32-36. 2 Dumont, Y., Fornier, A., St-Pierre, S., Schwartz, T.W. and Quirion, R., Differential distribution of neuropeptide Y1 and Y2 receptors in the rat brain, Eur. J. Pharmacol., 191 (1990) 501-503. 3 Dumont, Y., Fournier, A., St-Pierre, S. and Quirion, R., Comparative characterization and autoradiographic distribution of neuropeptide Y receptor subtypes in the rat brain, J. Neurosci., 13 (1993) 73-86. 4 Flood, J.F., Hernandez, E.N. and Morley, J.E., Modulation of memory processing by neuropeptide Y, Brain Res., 421 (1987) 280-290. 5 Fuhlendorff, J., Gether, U., Aakerland, L., Langeland-Johansen, N., Thogersen, H., Melberg, S.G., Olsen, U.B., Thastrup, O. and Schwartz, T.W., [Leu 31 ,Pro 34 ]Neuropeptide Y: a specific YI receptor agonist, Proc. Natl. Acad. Sci. USA, 87 (1990) 182-186.

6 Fuhlendorff, J., Langeland, N., Melberg, S.G., Thogersen, H. and Schwartz, T.W., The antiparallel pancreatic polypeptide fold in the binding of neuropeptide Y to Yl and Y2 receptors. J. Biol. Chem., 265 (1990) 11706-11712. 7 Herzog, H., Hort, Y.J., Ball, H.J., Hoyer, G., Shine, J. and Selbie, L.A., Cloned human neuropeptide Y receptor couples to two different second messenger systems, Proc. Natl. Acad. Sci. USA, 89 (1992) 5794-5798. 8 Inui, A., Sano, K., Miura, M., Hirosue, Y., Wakajima, M., Okita, M., Baba, S. and Kasuga, M., Evidence for further heterogeneity of the receptors for neuropeptide Y and peptide YY in tumor cell lines derived from neural crest, Endocrinology 131 (1992) 2090-2096. 9 Jolicoeur, F.B., Michaud, J.-N., Menard, D. and Fornier, A., In vivo structure activity study supports the existence of heterogeneous neuropeptide Y receptors, Brain Res. Bull., 26 (1991) 309-311. 10 Krause, J., Eva, C., Seeberg, P.H. and Sprengel, R., Neuropeptide Y: subtype pharmacology of a recombinantly expressed neuropeptide receptor, Mol. Pharmacol., 41 (1992) 817-821. 11 Larhammar, D., Bloomquist, A.G., Yee, F., Jazin, E., Yoo, H. and Wahlestedt, C., Cloning and functional expression of a human neuropeptide Y / peptide YY receptor of the YI type, J. Biol. Chem., 267 (1992) 10935-10938. 12 Lundberg, J.M., Terenius, L., H6kfelt, T., Martling, C.-R., Tatemoto, K., Miatt, V., Polak, J., Bloom, S. and Goldstein, M., Neuropeptide Y (NPY)-like immunoreactivity in peripheral noradrenergic neurons and effects of NPY on sympathetic function, Acta PhysioL Scand., 116 (1982) 477-790. 13 Lundberg, J.M., Hems6n, A., Rudehill, A., H~irfstrand, O., Larsson, A., Sollevi, A., Saria, A. H6kfelt, T., Fuxe, K. and Fredholm, B.B., (1988) Neuropeptide Y- and a-adrenergic receptors in pig

38 TABLE II

Specific binding of 100 pM [1251]PYY to sections of rat brain in the presence of either [LeuSl, ProS4]NpY or NPY13_s6 Results are expressed as fmol bound/mg protein. Mean +- S.E.M of data obtained from brains of six animals. Experiments were carried out in duplicate.

Brain region

Specific bound

Specific bound Specific bound + Yt agonist + Ye agonist

Frontal cortex layers I-III 10.3+_2.0 0.7+_0.1 layer IV 4.0+-0.2 0.2+-0.1 layers V-VI 2.2+-0.2 0.2+_0.1 Striatum 1.7+_0.2 0.64+_0.3 Globus pallidus 0.7+_0.1 0.49+_0.1 Nucleus accumbens 3.2+_0.4 0.69+_0.1 Olfactory tubercle 4.6 +_0.4 0.33 _+0.1 Corpus callosum 0.5+-0.1 0.48_+0.0 Thalamus Ventromedial nucleus 5.9 +- 1.4 0.6 + 0.3 Centromedial nucleus 6.1+-0.7 0.4+_0.1 Hypothalamus Medial preoptic nucleus 3.6 +-0.4 1.9 + 0.5 Lateral hyothalamic N. 3.5+_0.4 2.2+_0.2 Ventromedial nucleus 4.1+0.4 2.0+-0.4 Arcuate nucleus 5.8+-0.5 3.0+_0.4 Amygdala Basolateral nucleus 4.2 +_0.8 3.9 +_0.4 Basomedial nucleus 5.1+_0.4 4.3±0.2 Hippocampus Pyramidal layer C A l - : 11.5+_0.8 9.0+_0.5 Pyramidal layer CA3- 4 16.3+_1.1 13.8+_0.3 Dentate gyrus 4.4+_0.5 0.4+0.3 Superior colliculus Superficial layers 4.7±0.5 2.6+_0.3 Deep layers 3.2+_0.2 1.7±0.3 Inferior colliculus 7.2+_0.7 0.2+_0.0 Substantia nigra 3.6+_0.2 3.5+_0.2 Ventral tegmental area 4.7+-0.3 1.6+-0.5 Periaqueductal grey 3.4 +_1.3 1.6 ± 0.5 Lateral parabrachial N. 3.8+_0.5 2.4+_0.3 Pontine reticular area 2.5+_0.2 1 . 9 + _ 0 . 1 Locus coeruleus 5.8+_0.2 3.6+_0.4 Cerebellum 0.6+_0.1 0.4_+0.1

8.0___0.7 3.4___0.1 2.0+_0.2 0.7+_0.2 0.1___0.1 2.6+_0.2 2.9 +-0.3 0.2_+0.1 4.2 +-0.3 4.3___0.3 0.2 +-0.3 0.3±0.1 1.4±0.2 0.6±0.2 0.6 ± 0.2 0.4±0.1 1.1±0.3 1.6±0.6 2.9±0.5 2.0_+0.1 1.0±0.5 6.0+_ 1.2 1.2_+0.2 1.5+_0.1 1.6 +_0.5 1.1 ±0.0 0.2±0.1 0.4±0.2 0.2+-0.1

spleen: localization, binding characteristics, cyclic AMP effects and functional responses in control and denervated animals, Neuroscience, 24 (1988) 659-668. 14 Lynch, D.R., Walker, M.W., Miller, R.J. and Snyder S.N., Neuropeptide Y receptor binding sites in rat brain: differential autoradiographic localizations witht25I-peptide YY and 125I-neuropeptide Y imply receptor heterogeneity, J. Neurosci., 9 (1989) 2607-2619. 15 Martel, J.-C., St-Pierre, S., B6dard, P.J. and Quirion, R., Comparison of [laSI]Bolton-Hunter neuropeptide Y binding sites in the forebrain of various mammalian species, Brain Res., 419 (1987) 403-407. 16 Martel, J.-C., Fournier, A., St-Pierre, S. and Quirion, R., Quantitative autoradiographic distribution of [125I]Bolton-Hunter neuropeptide Y receptor binding sites in rat brain. Comparison with [125I]peptide YY receptor sites, Neuroscience, 36 (1990) 255-283. 17 Modin, A., Pernow, J. and Lundberg, J.M., Evidence for two neuropeptide Y receptors mediating vasoconstriction, Eur. J. Pharmacol., 203 (1991) 165-171. 18 Paxinos G. and Watson C., The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1982. 19 Paxinos, G.,The Human Nervous System, Academic Press, New York, 1990. 20 Rimland, J., Xin, W., Sweetnam, P., Saijoh, K., Nestler, E.J. and Duman, R.S., Sequence and expression of a neuropeptide Y receptor, Mol. PharmacoL, 40 (1992) 869-875. 21 Rioux, F., Bachelard, J.C., Martel, J.C. and St-Pierre, S., The vasoconstrictor effect of neuropeptide Y and related peptides in the isolated guinea-pig heart, Peptides, 7 (1986) 27-32. 22 Sheikh, S.P., Hakanson, R. and Schwartz, T.W., Yt and Y2 receptors for neuropeptide Y, FEBS Lett., 245 (1989) 209-214. 23 Tatemoto, K., Neuropeptide Y: complete amino acid sequence of the brain peptide, Proc. NatL Acad. Sci. USA, 79 (1982) 54855489. 24 Vallejo, M., Carter, D.A., Biswas, S., and Lightman S.L., Neuropeptide Y alters monoamine turnover in rat brain. Neurosci. Lett., 73 (1987) 155-160. 25 Wahlestedt, C., Yanaihara, N. and Hakanson, R., Evidence for different pre- and post-junctional receptors for neuropeptide Y and related peptides, Regul. Peptides, 13 (1986) 307-318. 26 Walker, M.W. and Miller, R.J.,125I-Neuropeptide Y and lZSlpeptide YY bind to multiple receptor sites in rat brain, Mol. PharmacoL, 34 (1988) 779-792. 27 Widdowson, P.S. and Halaris, A.E., A comparison of the binding of [3H]proprionyl-neuropeptide Y to rat and human frontal cortical membranes, J. Neurochem., 55 (1990)956-962.