Immunohistochemical localization of the neuropeptide Y Y1 receptor in rat central nervous system

Brain Research 889 (2001) 23–37 www.elsevier.com / locate / bres

Research report

Immunohistochemical localization of the neuropeptide Y Y1 receptor in rat central nervous system a a b b, Keisuke Migita , Arthur D. Loewy , Triprayar V. Ramabhadran , James E. Krause *, b Stephen M. Waters a

Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 63110, USA b Department of Biochemistry and Molecular Biology, Neurogen Corporation, Branford, CT 06405, USA Accepted 10 October 2000

Abstract The diverse effects of neuropeptide Y (NPY) are mediated through interaction with G-protein coupled receptors. Pharmacological analysis suggests the Y1 receptor mediates several of NPY’s central and peripheral actions. We sought to determine the distribution of Y1 protein throughout the rat central nervous system by means of indirect immunofluorescence using the tyramide signal amplification method and a novel, amino terminally-directed Y1 antisera. This antisera was verified as specific for Y1 by solution-phase competition ELISA, Western blot and in situ blocking experiments. High concentrations of Y1 immunoreactivity were found in the claustrum, piriform cortex (superficial layer), arcuate hypothalamic nucleus, interpeduncular nucleus, paratrigeminal nucleus, and lamina II of the spinal trigeminal nucleus and entire spinal cord. Moderate levels of Y1 immunoreactivity were found the in the main olfactory bulb, dorsomedial part of suprachiasmatic nucleus, paraventricular hypothalamic nucleus, ventral nucleus of lateral lemniscus, pontine nuclei, mesencephalic trigeminal nucleus, external cuneate nucleus, area postrema, and nucleus tractus solitarius. Low levels of Y1 immunostaining were distributed widely throughout layers II–III of the cerebral cortex (i.e., orbital, cingulate, frontal, parietal, insular, and temporal regions), nucleus accumbens core, amygdalohippocampal and amygdalopiriform areas, dentate gyrus, CA1 and CA2 fields of hippocampus, principal and oral divisions of the spinal trigeminal nucleus, islands of Calleja and presubiculum. These findings are discussed with

Abbreviations: 1–10, Spinal cord layers; III, Oculomotor nucleus; VII, Facial nerve or its root; XII, Hypoglossal nucleus; A5, A5 noradrenaline cell group; ac, Anterior commissure; AcbC, Accumbens nucleus, core; AcbSh, Accumbens nucleus, shell; AD, Anterodorsal thalamic nucleus; ADP, Anterodorsal preoptic nucleus; AHA, Anterior hypothalamic area, anterior part; AHiAL, Amygdalohippocampal area, anterolateral part; AHiPM, Amygdalohippocampal area, posteromedial part; AON, Anterior olfactory nucleus; AOP, Anterior olfactory nucleus, posterior part; AP, Area postrema; APir, Amygdalopiriform transition area; Arc, Arcuate hypothalamic nucleus; ATg, Anterior tegmental nucleus; AVPe, Anteroventral periventricular nucleus; BAOT, Bed nucleus of the accessory olfactory tract; Bar, Barrington’s nucleus; BLA, Basolateral amygdaloid nucleus, anterior part; CA1, Field CA1 of hippocampus; CA2, Field CA2 of hippocampus; CA3, Field CA3 of hippocampus; CeA, Central amygdaloid nucleus; CeCv, Central cervical nucleus; CL, Claustrum; cp, Cerebral peduncle, basal part; CPu, Caudate putamen; Cu, Cuneate nucleus; DC, Dorsal cochlear nucleus; DG, Dentate gyrus; DMH, Dorsomedial hypothalamic nucleus; DMX, Dorsal motor nucleus of vagus; DR, Dorsal raphe nucleus; ECu, External cuneate nucleus; EPl, External plexiform layer of the olfactory bulb; f, Fornix; FN, Facial nucleus; Gl, Glomerular layer of the olfactory bulb; GP, Globus pallidus; Gr, Gracile nucleus; ic, Internal capsule; ICj, Islands of Calleja; icp, Inferior cerebellar peduncle (restiform body); ION, Inferior olive nucleus; IP, Interpeduncular nucleus; IPI, Interpeduncular nucleus, intermediate subnucleus; IPR, Interpeduncular nucleus, rostral subnucleus; LatC, Lateral cervical nucleus; LC, Locus coeruleus; LGN, Lateral geniculate nucleus; LOT, Nucleus of the lateral olfactory tract; LPGi, Lateral paragigantocellular nucleus; LRt, Lateral reticular nucleus; LRtPC, Lateral reticular nucleus, parvicellular part; LS, Lateral septal nucleus; LSN, Lateral spinal nucleus; LV, Lateral vestibular nucleus; ME, Median eminence; Me5, Mesencephalic trigeminal nucleus; MGN, Medial geniculate nucleus; ml, Medial lemniscus; Mo5, Motor trigeminal nucleus; MVe, Medial vestibular nucleus; NA, Nucleus ambiguus; NTS, Nucleus of the solitary tract; OP, Optic nerve layer of the superior colliculus; ox, Optic chiasm; Pa5, Paratrigeminal nucleus; PAG, Periaqueductal gray; PB, Parabrachial nucleus; PDTg, Posterodorsal tegmental nucleus; PF, Parafascicular thalamic nucleus; Pir, Piriform cortex; Pn, Pontine nuclei; PO, Periolivary nucleus; Pr, Prepositus nucleus; Pr5, Principal sensory trigeminal nucleus; PrS, Presubiculum; PVN, Paraventricular thalamic nucleus; RCh, Retrochiasmatic area; RN, Red nucleus; RPC, Red nucleus, parvicellular part; Rt, Reticular thalamic nucleus; RVL, Rostroventrolateral reticular nucleus; SCN, Suprachiasmatic nucleus; SNR, Substantia nigra, reticular part; SO, Superior olivary nucleus; SON, Supraoptic nucleus; Sp5, Spinal trigeminal nucleus; Sp5C, Spinal trigeminal nucleus, caudal part; Sp5O, Spinal trigeminal nucleus, oral part; SpVe, Spinal vestibular nucleus; STh, Subthalamic nucleus; SuG, Superficial gray layer of the superior colliculus; Tz, Nucleus of the trapezoid body; VA, Ventral anterior thalamic nucleus; VCA, Ventral cochlear nucleus, anterior part; VCP, Ventral cochlear nucleus, posterior part; VLL, Ventral nucleus of the lateral lemniscus; VMH, Ventromedial thalamic nucleus; VP, Ventral pallidum; VPM, Ventral posteromedial thalamic nucleus; Y, Nucleus Y *Corresponding author. Tel.: 11-203-488-8201, ext. 3029; fax: 11-203-483-8317. E-mail address: [email protected] (J.E. Krause). 0006-8993 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )03092-4

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reference to previously reported receptor autoradiography, immunohistochemistry and mRNA analyses to further support the role of Y1 in NPY-mediated biology.  2001 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Regional localization of receptors and transmitters Keywords: Hypothalamus; Suprachiasmatic nucleus; Spinal cord; Pain

1. Introduction Neuropeptide Y (NPY) is a 36 amino acid peptide sharing strong sequence homology with pancreatic polypeptide and peptide YY. NPY is widely and abundantly expressed in CNS [2,11] and mediates many physiological actions. NPY is associated with induction of food intake [12,38,66,70], blood pressure regulation [27,50], anxiolysis [65,74], seizure threshold [18,41], memory enhancement [5,73], and spinal analgesia to thermal stimuli [80,81]. To date, six mammalian, G-protein coupled, NPY receptors have been described. In rat, four NPY receptors have been cloned including Y1 [19], Y2 [71], Y4 [85] and Y5 [20,32]. Each of these receptor subtypes are further defined by their distinct pharmacology. Y1 preferentially binds NPY, peptide YY and [Leu 31 ,Pro 34 ]-NPY with pancreatic polypeptide having much lower affinity [20]. NPY, peptide YY and short C-terminal fragments of NPY including NPY 13 – 36 and NPY 18 – 36 have the greatest affinity for Y2 [21]. Y4 selectively binds pancreatic polypeptide with NPY or peptide YY showing no detectable affinity for this receptor [85]. NPY, peptide YY, pancreatic polypeptide, [Leu 31 ,Pro 34 ]-NPY, [D-Trp 32 ]-NPY and long Cterminal fragments including NPY 2 – 36 and peptide YY 3 – 36 all demonstrate high affinity for Y5 [20]. Y1 is perhaps the most widely studied NPY receptor and is considered to be involved with many of the central and peripheral effects of NPY. Injection of NPY i.c.v. produces a rapid increase of food intake, which is attenuated by the Y1-selective antagonist BIBP3226 [40,57]. Additionally, while Y1 knockout mice show only modest changes in NPY-induced feeding behavior [39,61] marked reductions are observed in fasted knockout animals [61]. In hypothalamus, Y1 mRNA is detected by in situ hybridization [10,58], and the presence of Y1 protein is suggested by receptor autoradiography [16] and confirmed by immunohistochemistry [87], further supporting a role for Y1 in hypothalamic, NPY-mediated biology. In the basolateral nucleus of the amygdala, direct NPY injection decreases anxiety in behavioral tests [65]. This anxiolytic effect of NPY is blocked with coadministration of a Y1-selective antagonist [65] or lateral ventricle injection of Y1 antisense oligodeoxynucleotides [79]. Peripheral injection of NPY or [Pro 34 ]-NPY produces a marked increase in arterial pressure whereas NPY 2 – 36 , NPY 4 – 36 and NPY 11 – 36 are without effect suggesting a Y1-mediated response [26]. Y1 knockout mice confirm these results as to the impor-

tance of Y1 in the cardiovascular effects of NPY [61]. During kindling epileptogenesis, NPY receptor mRNAs, including Y1, are altered in several limbic regions [41]. Additionally, NPY peptide mRNA is increased in models of spinal nerve injury [48] and the mechanical hyperalgesia noted in sciatic nerve injury demonstrates Y1-like pharmacology [81]. Because Y1 mediates many NPY central and peripheral actions, it is of interest to determine the discrete localization of Y1 protein in regions associated with NPY biology. The objective of the present study was to use a specific Y1 amino terminally-directed antisera and immunohistochemistry to determine the distribution of Y1 protein in rat brain and spinal cord.

2. Materials and methods

2.1. Antisera generation and characterization A rat Y1 amino terminus peptide [SRVENYSVHYNVSENSPFLA, Y1 (7–26)] was synthesized (75% purity, Keck Foundation Biochemistry Resource Laboratory, Yale University) and coupled to maleimideactivated keyhole limpet hemocyanin (Pierce, Rockford, IL). Polyclonal antibodies were raised in rabbits (Cocalico Biologicals, Inc., Reamstown, PA) against the rat Y1 receptor conjugated peptide and purified using an immunoaffinity purification column (Pierce). Antisera specificity was evaluated by solution-phase competition enzymelinked immunosorbent assay (ELISA). The rat Y1 (7–26) peptide was coated onto Falcon 96 well Microtest tissue culture plates at 1 mg / well in 50 ml phosphate buffered saline (PBS), pH 7.4, by overnight incubation at 48C. The wells were blocked with 3% bovine serum albumin (BSA, Sigma), 0.02% sodium azide in PBS for 2 h at room temperature. Primary and secondary antibodies were diluted in PBS containing 3% BSA and 0.02% sodium azide. Incubation with Y1 antisera (1:2000 dilution) was for 2 h in the presence of 10 210 to 10 24 M of Y1 (7–26), rat Y5 (16–35, 75% purity, Keck Foundation Biochemistry Resource Laboratory), rat corticotrophin releasing factor 2a receptor [CRF2a (20–37), 75% purity, Peptide Chemistry, Washington University], rat neurokinin-1 receptor [NK1 (376–407), 85% purity, Washington University], or human neurokinin-3 receptor [NK3 (434–465), 80% purity, Washington University] peptides as competitor. Incubation

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with goat anti-rabbit IgG coupled to alkaline phosphatase (Life Technologies, Baltimore, MD), at a dilution of 1:500, was for 1 h at 378C. p-Nitrophenyl phosphate disodium (Sigma), 1 mg / ml in 10 mM diethanolamine and 0.5 mM MgCl 2 , was incubated at 378C for 30 min as a substrate and read at 405 nm absorbance for detection. Antisera specificity also was determined by Western blot. Cell membranes (2.5 mg) derived from rat Y1, rat Y2, human Y4, rat Y5 and untransfected baculovirus-infected Sf9 cells were run on a 8–16% Tris–glycine polyacrylamide gel (Novex, Invitrogen, Carlsbad, CA) under denaturing conditions. Membranes were incubated in sample buffer at 458C prior to loading to limit receptor aggregation. The gel was blotted onto nitrocellulose and Ponceau S stain was used to assess sample loading and transfer efficiency. Membranes were blocked with 5% non-fat dry milk for 60 min then incubated overnight at 48C with a 1:500 dilution of Y1 antisera in TBST (10 mM Tris, 150 mM NaCl and 0.05% Tween-20). Membranes were washed 3 times with TBST and incubated with a 1:2,000 dilution of donkey anti-rabbit IgG coupled to horseradish peroxidase (Amersham, Chicago, IL) for 90 min at RT. Membranes were washed 4 times with TBST and developed using a ECL1Plus Western blotting detection system (Amersham).

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Method, Product NEL 700; NEN Life Science Products, Boston, MA) for 15 min. Subsequently, sections were washed in KPBS, reacted with Cy3-conjugated streptavidin (1:200, Jackson) for 2 h, washed again, mounted on gelatin-coated glass slides, dried, and coverslipped with an anti-fade glycerol solution. Sections were examined by epifluorescence optics and each section from a 1-in-5 series of the entire brain from four animals was photographed with a SIT digital camera (Dage –MTI SIT 68, Michigan City, IN). Images were montaged using the Adobe Photoshop 5.5 program. Gray scale images were converted to a range of pseudo-colors. Color printouts were made, and each region of the brain given a score where red represented the heavy labeling (13), orange appeared as medium labeling (12), and yellow signified weak labeling (11). Using the information obtained in these color printouts, we then constructed the staining patterns presented in the line drawings shown in Fig. 5. These drawings were taken from the rat atlas published by Paxinos and Watson [60] with slight modifications.

3. Results

3.1. Characterization of Y1 antisera 2.2. Immunohistochemistry The research described in this report was reviewed and approved by the Washington University School of Medicine Animal Care Committees and conformed to NIH guidelines. Sprague–Dawley rats (n510, female, 250 gm, Harlan Labs) were anesthetized with sodium pentobarbital (50 mg / kg, i.p.) and perfused through the heart with saline, followed by 4% paraformaldehyde made in 0.1 M sodium phosphate buffer (pH57.4). The brain and spinal cord was removed, stored in fixative for 3–7 days, and transverse sections were cut in at 50 mm on a freezing microtome. The sections were collected in plastic tissue culture plates containing 0.1 M sodium phosphate buffer with 0.1% sodium azide. A 1-in-5 series through the brains from six of these animals were used to obtain a complete series of sections stained for Y1. The complete spinal cord from four rats was processed for immunohistochemistry. The brains from the other four rats were used to test the specificity of the antisera. Sections were incubated overnight in rabbit anti-Y1 (1:2000) diluted in 5% donkey serum and in potassium buffered saline (KPBS, pH57.4). Sections were then washed in KPBS, transferred to biotinylated donkey antirabbit (1:100; Jackson ImmunoResearch Lab, West Grove, PA) for 3 h. Next, the sections were washed in KPBS, and transferred to peroxidase-conjugated streptavidin (1:500; Jackson Lab) for 2 h, washed again, and placed into a solution of biotinylated tyramide (1:300, TSA Indirect

Immunoaffinity purified polyclonal antisera raised against the amino terminus of rat Y1 was evaluated for antisera specificity by solution-phase competition ELISA (Fig. 1). Antisera binding was blocked only with the Y1 (7–26) peptide as competitor. Inhibition neared 90% at a concentration of 10 mM immunizing peptide. No apparent

Fig. 1. Solution-phase competition ELISA for Y1 antisera. Y1 antisera was incubated in the presence of the immunizing peptide rat Y1 (7–26), rat Y5 (16–35), rat corticotrophin releasing factor 2a receptor [CRF2a (20–37)], rat neurokinin-1 receptor [NK1 (376–407)] or human neurokinin-3 receptor [NK3 (434–465)] peptides as competitor for solidphase Y1 (7–26) peptide.

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competition was observed using Y5 (16–35, related NPY receptor N-terminus control), CRF2a (20–37, irrelevant N-terminus control), NK1 (376–407, irrelevant C-terminus control) or NK3 (434–465, irrelevant C-terminus control) peptides as competitor up to a concentration of 10 24 M. Antisera specificity also was determined by Western blot (Fig. 2). Y1 antisera only reacted with rat Y1 baculovirusinfected Sf9 cell membranes with no apparent signal in rat Y2, human Y4, rat Y5 or untransfected membranes. Two major bands (40–43 kD and 72–74 kD) were observed in Y1 membranes. Native rat Y1 is 44.1 kD and contains three potential N-linked glycosylation sites. Therefore, the lower band may represent native Y1 with the larger band corresponding to glycosylated forms of Y1. The Y1 signal was completely blocked by coincubation of Y1 antisera with 10 mM immunizing peptide (data not shown).

3.2. Immunohistochemistry

Fig. 2. Western blot analysis of Y1 antisera specificity. Membranes (2.5 mg) from Sf9 cells infected with either rat Y1, rat Y2, human Y4, rat Y5 or untransfected (WT) were subjected to 8–16% Tris–glycine polyacrylamide gel electrophoresis under denaturing conditions, blotted onto nitrocellulose and analyzed as described in Materials and methods. Positions of prestained weight standards (M) are indicated on the left in kD.

Antisera specificity was further tested in situ by coincubation of primary antisera with immunizing peptide. Sections containing the arcuate hypothalamic nucleus were reacted with the same antisera solution described above and compared in parallel fashion with antisera solution in the presence of 1 mM of Y1 (7–26). As shown in Fig. 3, the addition of this peptide prevented Y1 staining in the arcuate region. A second series of blockade experiments were performed on brainstem sections containing the caudal division of the spinal trigeminal nucleus; addition of the Y1 peptide to the antisera solution blocked the immunostaining. Additionally, controls were performed in which sections were reacted with the omission of the primary antiserum. False-positive staining was not found. Immunoreactive staining for Y1 was observed in many regions throughout the brain and spinal cord. In all regions, immunoreactivity was primarily distributed throughout the neuropil with occasional cell body staining observed. This was most evident in arcuate hypothalamic nucleus due to the high density of receptors and packaging of labeled

Fig. 3. In situ specificity of Y1 antisera. (A) Y1 immunoreactivity is present in the arcuate hypothalamic nucleus and median eminence; and (B) Block of Y1 immunoreactivity by Y1 (7–26) immunizing peptide (1 mM).

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neurons (Fig. 3). However, at other sites such as dorsal horn, it was not possible to resolve individual perikarya because of the small size and dense packing of these neurons. Fig. 4 shows examples of three different levels of immunostaining as described in the Materials and methods section and mapped onto coronal sections in Fig. 5. In the telencephalon, dense concentrations of Y1 immunoreactivity were found throughout the claustrum (Fig. 5C– G) and piriform cortex (superficial layer) (Fig. 5B–E). Moderate levels of Y1 were identified in the glomerular, external and internal plexiform layers of the olfactory bulb (Fig. 5A). Weak levels of Y1 immunoreactivity were found in layers II–III of the cerebral cortex (Fig. 5B–K). Many cortical areas were labeled including the orbital, cingulate,

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frontal, parietal, insular, and temporal regions. In addition, diffuse labeling was found in the presubiculum (Fig. 5K). In the basal forebrain region, weak labeling was found in the anterior olfactory nucleus (Fig. 5B,C), shell and core of the nucleus accumbens (Fig. 5D), ventrolateral part of the caudate-putamen (Fig. 5E), ventral pallidum (Fig. 5E), islands of Calleja (Fig. 5E), dentate gyrus, CA1 and CA2 hippocampal field (Fig. 5G–I). In hypothalamus, dense Y1 labeling was seen in the arcuate hypothalamic nucleus and median eminence (Fig. 3 and Fig. 5H,I). Moderate labeling was found in the dorsomedial suprachiasmatic nucleus (Figs. 5F and 6A). Weak levels of immunoreactivity were found in the medial parvicellular part of the paraventricular hypothalamic nucleus (Fig. 5G). In the thalamus, weak labeling was detected in anterodorsal and reticular thalamic nuclei. In midbrain, the interpeduncular nucleus (Fig. 5J) contained heavy labeling, while moderate labeling was found in the oculomotor nucleus (Fig. 5J). The superficial layer of the superior colliculus (Fig. 5J,K) and the reticular part of the substantia nigra (Fig. 5J) contained weak amounts of Y1 immunoreactivity. No immunoreactivity was detected in the periaqueductal gray matter (Fig. 5J– K). In the pons, moderate labeling was detected in the ventral nucleus of the lateral lemniscus (Fig. 5K) and pontine nucleus (Fig. 5K). No labeling was found in the parabrachial nucleus, locus coeruleus, or Barrington’s nucleus, although a moderate density of labeling was observed in the mesencephalic trigeminal nucleus (Fig. 5L). Weak labeling was present in the principal trigeminal nucleus and superior olivary complex, with the trapezoid nucleus showing a moderate concentration of Y1 immunoreactivity (Fig. 5L). In the medulla oblongata, a dense concentration of Y1 labeling was found in layer II of the caudal spinal trigeminal nucleus (Fig. 5P,Q) and paratrigeminal nucleus (Fig. 5O). Moderate levels of immunoreactivity were found in the ventral cochlear nucleus (Fig. 5L,M), inferior olive (Fig. 5N,O), dorsal part of the gracile nucleus (Figs. 5P and 6B), external cuneate nucleus (Figs. 5O and 6B), and in the interstitial portion of the nucleus tractus solitarius (Figs. 5O and 6B). Diffuse labeling was found in the vestibular nuclei, rostral part of the spinal trigeminal nucleus, lateral reticular nucleus, and dorsal column nuclei (Fig. 5N,O). Throughout the spinal cord, strong labeling was observed in lamina II (Fig. 7).

4. Discussion

Fig. 4. Examples of the three different epifluorescent staining levels. (A) Immunoreactivity present in the red nucleus, parvicellular part, representing weak or 11 labeling; (B) Immunoreactivity in oculomotor nucleus representing moderate or 12 labeling; and (C) Immunoreactivity in the claustrum representing high or 13 labeling.

The diverse functions of NPY in the brain and spinal cord are mediated through interaction with multiple Gprotein coupled receptors. Pharmacological studies suggest that Y1 plays a significant role in many NPY-associated effects. The presence of Y1 mRNA and putative binding sites in several NPY-rich regions support these observations. Previous analyses demonstrate the presence of NPY1 immunoreactivity in regions of rat CNS using a carboxy

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Fig. 5. Y1 receptor labeling throughout the rat brain presented in line drawings modified from Paxinos and Watson [60]. Three different densities of immunoreactivity were found and are presented. Weak or 11, moderate or 12, high or 13 (see Fig. 4).

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Fig. 5. (continued)

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Fig. 5. (continued)

terminally-directed rabbit antisera [87], second extracellular loop-directed chicken antisera and third extracellular loop-directed rabbit antisera [62]. The goal of the present study was to determine the distribution of Y1 protein throughout the entire brain and spinal cord using a specific Y1 amino terminally-directed antisera and the highly sensitive tyramide signal amplification immunohistochemical method [64,75]. The antisera used in the present experiments was shown to be specific for Y1 in Western blotting experiments as no cross reactivity was observed in Y2, Y4 or Y5 membranes. Additionally, Y1 signal was abolished by coincubation with Y1 immunizing peptide in both ELISA and immunohistochemistry experiments, further demonstrating Y1 specificity.

4.1. Telencephalon NPY is associated with many telencephalon effects including memory enhancement (at low doses) [73], modulation of seizure threshold [37,41,54] and feeding behavior [29]. These effects are presumably mediated through NPY receptors in cortex, hippocampus, amygdala and nucleus accumbens. Previous work using a radioactive non-peptide Y1 antagonist, BIBP3226 [17], and Y1 agonist, [Leu 31 ,Pro 34 ]-porcine peptide YY (pPYY), to localize Y1 binding sites in rat brain showed high concentrations of binding to superficial laminae of cerebral cortex [17]. However, using an enhanced immunohistochemical localization method in the present experiments, we found

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Fig. 6. (A) Y1 receptor immunoreactivity in the dorsomedial part of the suprachiasmatic nucleus (SCN); and (B) Y1 immunoreactivity in the dorsomedial medulla oblongata. Moderate labeling was found in the area postrema (AP), weak amounts in the interstitial part of the nucleus tractus solitarius (NTS), gracile (Gr) and external cuneate (ECu) nuclei, and dense labeling the paratrigeminal nucleus (Pa5). Very weak labeling was found in the spinal trigeminal nucleus (SP5), and none in the dorsal vagal nucleus (DMX) and hypoglossal nucleus (XII).

relatively weak labeling throughout layers II and III of cerebral cortex. In hippocampus, we found low to moderate levels of Y1 immunoreactivity in CA1, CA2 and dentate gyrus with no labeling in CA3. Y1 immunoreactivity also was observed in the amygdalohippocampal and amygdalopiriform areas. Several studies suggest a role for NPY in seizure modulation mediated through receptors in cortex, hippocampus and amygdala [37,41,54]. Interestingly, in hippocampus and amygdala, Y1 mRNA is decreased during kindling epileptogenesis whereas NPY, Y2

and Y5 mRNAs are increased in these experiments [41]. Therefore, it has been suggested that activation of Y1 in dentate gyrus produces proconvulsant activity whereas Y2 and Y5 mediate the anticonvulsant properties of NPY [41,78]. This hypothesis is supported by results showing that intrahippocampal injection of a Y1 antagonist, BIBP3226, reduces kainate-induced EEG seizures in rats and this effect is reversed by [Leu 31 ,Pro 34 ]-NPY [78]. In nucleus accumbens, our results concur with Pickel et al. [62] who show higher Y1 immunoreactivity in the core

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Fig. 7. (A–D) Examples of Y1 receptor immunoreactivity found in the substantia gelatinosa of the spinal cord at cervical (A), thoracic (B), lumbar (C), and sacral (D) levels.

versus the shell region. NPY-mediated modulation of nucleus accumbens core outflow to the ventral pallidum could support a non-hypothalamic component of the NPY feeding response via regulation of motor function [30].

Interestingly, the nucleus accumbens is devoid of Y1 mRNA [58]. This suggests that these Y1-containing terminals are projections from more distant sites such as the ventral tegmental area, which contains Y1 mRNA [58].

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We also found Y1 immunoreactivity in several other basal forebrain areas such as the claustrum, islands of Calleja, ventral pallidum, ventral part of the caudateputamen and basal nucleus of the optic tract in correlation with earlier binding studies [17,43]. Conversely, dense Y1 immunoreactivity was found in the superficial layer of piriform cortex while mRNA studies show more extensive labeling in this region (e.g., [41]). Similarly, binding studies reveal high concentrations of Y1 binding in septum [17,43], however, this area was devoid of Y1 immunoreactivity and is negative for mRNA by in situ hybridization [43]. Differences noted between autoradiography and immunohistochemistry are likely due to the nonspecificity of some ligands used for autoradiography analysis. For example, [Leu 31 ,Pro 34 ]-PYY has high affinity for both Y1 and Y5 [23]. Therefore, differences noted between immunoreactivity and autoradiography would suggest the presence of other NPY receptor subtypes in those regions.

4.2. Diencephalon The hypothalamic control of feeding is one of the major functions associated with NPY. Injection of NPY i.c.v. [12,86] or in discrete subregions of the hypothalamus including the paraventricular nucleus, ventromedial hypothalamus and lateral hypothalamus [69], results in marked increases in food intake. Y1 mRNA is highly expressed in the arcuate nucleus and supraoptic nucleus with moderate expression in paraventricular nucleus, ventromedial hypothalamus and lateral hypothalamus [58]. In agreement with previous autoradiography studies [8,17,43], dense concentrations of Y1 were detected in arcuate hypothalamic nucleus with moderate levels in the medial parvocellular division of the paraventricular nucleus. In arcuate nucleus, Y1 is expressed on proopiomelanocortin (POMC) cell bodies and inhibits POMC neuronal activity [7]. Moreover, POMC and NPY neurons in arcuate nucleus project to the paraventricular nucleus [6] and these peptides are functional antagonists for feeding, possibly through interactions at GABAergic neurons [14]. While these GABAergic neurons demonstrate Y1-like sensitivity [63], the presence of Y5 in the paraventricular nucleus [20] suggests that the hypothalamic feeding effects of NPY are not exclusive to Y1. Indeed, receptor knockouts for Y1 [39,61], Y2 [55] and Y5 [49] all show alterations in food intake and body weight. Y1 immunoreactivity was also detected in the suprachiasmatic nucleus (SCN). NPY infusions into the SCN cause phase shifts in circadian rhythms [1,52,68], which can be blocked by local injections of bicuculline, a GABA–A receptor antagonist [33]. When muscimol, a GABA-A agonist, is injected into the SCN, it causes a reduction in the phase-delaying effect of light on somatomotor activity [22]. NPY exerts both a phase shifting and inhibitory effect on SCN neurons, but has two different effective concentration levels [25]. Concentra-

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tion-response curves show that NPY produces phase shifting at 54 nM while the EC 50 for inhibition of SCN neural firing is 113 nM [25]. Moreover, the phase-advancing effect of NPY is mediated by the Y2 receptor [24,25,34], which appears to be independent of its inhibitory action. In addition to Y1 and Y2, Y5 receptors are present in the SCN, but peptide agonists used to define this receptor system do not effect phase-shifting [25]. Y1 immunoreactivity was restricted to the dorsomedial part of the SCN, which is important in regulation of the autonomic nervous system [77]. The SCN receives an NPY input from a highly restricted part of the lateral geniculate thalamic nucleus, the intergeniculate leaflet [9,28,35,76]. This projection terminates mainly in the ventrolateral part of the SCN, the same area that receives an input from the retinal ganglion cells [53]. These retinal neurons (W-type) transmit luminosity information, providing an entraining input to the SCN that is critical for the timing of circadian functions. The reason for the apparent dichotomy between our findings showing that Y1 immunoreactivity is concentrated in the dorsomedial SCN, but NPY fibers are localized in the ventrolateral SCN is unclear. Dumont et al. [17] and Larsen et al. [43] were able to detect moderate levels of Y1 receptors throughout the thalamus but our findings revealed that Y1 immunoreactivity was restricted to only two thalamic regions (i.e., anterior dorsal and reticular thalamic nuclei). Larsen et al. [43] also detect Y1 receptors in the subfornical organ and median preoptic nucleus whereas we did not observe Y1 immunoreactivity. These disparities may result from the presence of additional receptor subtypes that bind [Leu 31 ,Pro 34 ]-PYY in those regions.

4.3. Mesencephalon In midbrain, dense concentrations of Y1 immunoreactivity were found in the interpeduncular nucleus, which agrees with the findings of Larsen et al. [43]. However, we found Y1 immunoreactivity in the pars reticulata of the substantia nigra, but not in the pars compacta region as reported by Larsen et al. [43]. Similarly, we found moderate concentrations of Y1 receptors in the oculomotor nucleus whereas binding studies did not show labeling in this region [43]. We failed to find any Y1 immunoreactivity in the periaqueductal gray matter, although Larsen et al. [43] reported this region to contain binding sites. In addition, weak Y1 immunoreactivity was found in the present study in the superficial layer of the superior colliculus and dense labeling in the ventral lateral lemniscus nucleus. Previous reports did not analyze these regions.

4.4. Metencephalon In the present study, we have demonstrated that Y1 receptors are localized in the interstitial region of the

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nucleus tractus solitarius (NTS). While NPY immunoreactivity is found throughout the NTS [45], the concentration of Y1 receptors in this NTS subnucleus is interesting because both the superior laryngeal and recurrent laryngeal nerves project to the interstitial as well as to the medial NTS regions [3,31,46,59]. Currently, there is no evidence to suggest that Y1 receptors subserve laryngeal functions. In rat, only a few nodose ganglion cells contain NPY [15,88], and none express Y1 receptors [88]. However, application of a Y1 peptide agonist to dispersed nodose neurons results in an increase in calcium currents in approximately 50% of these cells [82]. Other NPY expressing vagal sensory neurons that lie along the distal parts of the superior laryngeal and vagal nerve, such as found in the cat [72], may contribute to this system as well. Y1 immunoreactivity was found in the area postrema but not other circumventricular organs such as the subfornical organ (SFO) and the organum vasculosum of the lamina terminalis (OVLT). These findings are consistent with data obtained by autoradiographic binding [17,43,44]. The area postrema is implicated as a potential site affecting cardiovascular responses [4] while the SFO and OVLT function primarily as critical sites regulating body fluid homeostasis [51]. The functions of the Y1 receptors localized in the area postrema remain uncertain, but may be related to central mechanisms that regulate renal function particularly sodium excretion [13]. In this regard, it is interesting to note the predominant type of NPY receptor found in the vascular bed of the kidney is the Y1 type [47], which is situated postjunctionally and mediates vasoconstriction. Experiments by Nishida et al. [56] demonstrate in rabbits that the removal of the area postrema results in an impaired ability to suppress renal sympathetic nerve activity and sodium excretion following increased vascular salt loading. Perhaps a NPY hormonal feedback mechanism operates through Y1 receptors at both the area postrema and renal vascular system to maintain sodium balance. The paratrigeminal nucleus receives convergent visceral and somatic sensory information from the trigeminal, glossopharygneal, vagus, and cervical spinal nerves, which carry sensory information from orofacial, upper respiratory, and neck structures. Presumably the nucleus relays information related to noxious stimuli in these areas to the NTS for visceral reflex adjustments and to rostral sites such as the parabrachial nucleus and ventroposterior medial thalamus for sensory perception [67]. The functional significance of high concentrations of Y1 receptors in the paratrigeminal nucleus is unknown, but may mediate similar functions as the Y1 receptors found in lamina II region of the spinal trigeminal nucleus and spinal cord. These Y1 receptors may be part of a common nociceptive transducing mechanism, but experiments addressing this issue have not yet been reported (see below for further discussion). The Y1 receptor knockout mice may be an excellent model system to explore this possibility [42,61].

4.5. Spinal cord Throughout the entire length of the spinal cord, Y1 was concentrated in the substantia gelatinosa (lamina II). Zhang et al. [87] report Y1 on small dorsal root ganglion (DRG) neurons and in the lamina II of the spinal cord in rats. Y1 present on DRG neurons are postsynaptic receptors that may be affected by circulating levels of NPY produced by sympathetic neurons or the adrenal medulla. In lamina II, Y1 is present on somatostatin neurons [89], which may be part of an intrinsic dorsal horn circuit that modulates pain transmission. Intrathecal application of NPY causes antinociception, which is independent of opioid and alpha-2 adrenergic spinal mechanisms [84]. A Y1 / 5 agonist, [Leu 31 ,Pro 34 ]-NPY, given intrathecally to rats with intact sciatic nerves, facilitates the nociceptive flexor reflex test at low does, but at high doses, facilitation is followed by depression [83]. This suggests that Y1 may be responsible for the reflex depressive effect of NPY in intact animals [83]. When peripheral nerve injury or inflammation occurs, an upregulation of NPY and Y1 occurs in DRG and laminae II and III of the dorsal horn [36], suggesting a role for Y1 in pain modulation in response to injury.

4.6. Summary and conclusions The diverse actions of NPY including regulation of feeding, seizure threshold, blood pressure and analgesia are all likely mediated, in part, by Y1. The presence of Y1 protein in brain and spinal cord regions associated with NPY-mediated biology was confirmed by the present immunohistochemical analysis. The concurrent presence of mRNA and binding sites for additional NPY receptor subtypes in these regions suggests a complex regulation of NPY effects. While the generation of NPY receptor knockout animals are beginning to confirm receptor specific effects of NPY, the continued development of high affinity, specific agonists and antagonists for NPY receptor subtypes will be required for direct association of NPY effects and individual receptor subtypes.

Acknowledgements This work was supported in part by NIH grant HL25449 to A.D.L. The authors would like to thank Li Shao, Dr. Yuriko Suzuki, Jennifer Carrier, Andrzej Kieltyka and Dr. Robb Brodbeck for technical assistance.

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