J Pined Res 1993;15:53-58 Printed in the United States of Amerira--all rights reserved

Copyright 0 Munksgnard, 1993

Journal of Pineal Research ISSN 0742-3098

An immunohistochemical study of neuropeptide Y in the bovine Dineal gland I U

Phansuwan-Pujito P, Pramaulkijja S, Govitrapong P, Moller M. An immunohistochemical study of neuropeptide Y in the bovine pineal gland. J. Pineal Res. 1993:15:53-58.

Abstract: An immunohistochemical study of the bovine pineal gland was performed using rabbit polyclonal antibodies raised against neuropeptide Y (NPY) or against the C-terminal flanking peptide of proNPY (CPON) . A large number of NPYXPON-immunoreactive (IR) nerve fibers were demonstrated throughout bovine pineal gland. The IR-fibers were located in the capsule of the gland, usually piercing into the gland together with blood vessels. In the gland itself, the fibers were also located intraparenchymally between the pinealocytes. Within the rostra1 and caudal areas of the pineal stalk, NPY-IR fibers were also observed, and these fibers could be followed not only into the gland but also to the habenular and posterior commissures. The morphological localization of the NPY-IR nerve fibers in the bovine pineal gland indicate that the majority of fibers originate from the superior cervical ganglion. However, some fibers probably originate from the brain itself.

Introduction

The pineal gland is an important component of the brain photoneuroendocrine system [Goldman and Darrow , 19831. The gland synthesizes and releases melatonin as well as other possible functionally important indoles [PCvet et al., 19861. The melato- nin synthesis is primarily regulated by the noradren- ergic sympathetic system from superior cervical ganglion [Sugden, 19911. However, nerve fibers containing a number of peptides, originating from extrasympathetic sources, have been demonstrated to innervate the gland [Korf and Mgller, 1984; Ebadi et al., 1989; Mgller et al., 19911.

Neuropeptide Y (NPY), a naturally occurring neuropeptide originally isolated from the porcine brain [Tatemoto et al., 19821, consists of 36 amino acid residues with a C-terminal tyrosyl-amide. It is widely distributed within the central [Allen et al., 19831 and peripheral [Lundberg et al., 19821 ner- vous system including the pineal gland [Chronwall et al., 19851. By immunohistochemistry, immunor-

Pansiri Phansuwan-Pujito,' Sarita Pramaulkijja,* Piyarat Govitrapong,2 and Morten Mnlle? 'Department of Anatomy Faculty of Medicine, Srinakarinwirot University at Prasarnmit, Bangkok, Thailand; 'Neuro-and Behavioral Biology Center, Institute of Science and Technology for Development, Mahidol University at Salaya. Nakornpathom, Thailand; 31nstitute of Medical Anatomy, University of Copenhagen, The Panum Institute, Copenhagen, Denmark

Key words: neuropeptide Y-bovine-pineal gland-innervation

Address reprint requests to M. Msller, MD., Ph.D., Institute of Medical Anatomy, Department B, The Panum Institute, Blegdamsvej 3, DK-2200 Copenhagen, Denmark

Received December 14, 1992; accepted March 5, 1993.

eactive nerve fibers containing NPY have been detected earlier in the pineal gland of rat [Schon et al., 1985; Zhang et al., 19911, hamster [Schroder, 1986; Li and Welsh, 19911, Mongolian gerbil [Shiotani et al., 19861, guinea pig [Schroder and Vollrath, 19861, and sheep [Williams et al., 1989; Cozzi et al., 19921. In rodents most of the NPYergic nerve fibers in the superficial pineal gland originate from the superior cervical ganglion [Zhang et al., 19911. However, in the rat NPY-immunoreactive (IR) nerve fibers, located in the pineal stalk and the deep pineal gland, remain after removal of both superior cervical ganglia [Zhang et a]., 1991 1. In the sheep, the number of NPYergic fibers remaining after bilateral removal of the superior cervical ganglion is high [Cozzi et al., 19921, indicating a prominent extrasympathetic innervation of the gland in this species.

In the present investigation, we have been able to show the presence of an intense NPY-immunoreac- rive innervation of the pineal gland of bovine, a herbivorous animal, by using different antisera

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Phansuwan-Pujito et al.

raised against NPY or the C-terminal flanking peptide of proNPY (CPON).

Materials and methods

Antisera and peptides

The antiserum against the C-flanking peptide of proNPY (CPON) was purchased from Cambridge Research Biochemicals (Cambridge, UK). An an- tiserum (#8999) against NPY was raised in rabbits. This antiserum has been shown to react with ami- dated NPY, .36 and NPY,,_,, but not with NPY free acid [see Cozzi et al., 19921.

Animals

Pineal glands of bovines (Bos tauvus) were obtained from a slaughterhouse in Copenhagen. The glands connected to the epithalamus were dissected from brains 10-30 rnin after the animals were killed.

Fixation of tissues

The bovine pineal glands with the epithalamus were immediately immersion fixed in 4% paraformalde- hyde in 0.1 M NdK-phosphate buffer (KPB) (pH 7.4) at 4°C for 2 days. The glands were then cryoprotected in 20% sucrose in phosphate-buffered saline (KPBS) (pH 7.4) overnight and sectioned frontally and sagitally in a cryostat at a thickness of 40 pm and rinsed in KPBS.

lmmunohistochemical procedures

The immunohistochemical localization of NPY and CPON was performed on free floating sections by use of the streptavidin biotin-complex method. First, the sections were rinsed for 10 min in KPBS containing 0.25% bovine serum albumin (BSA) and 0.1% Triton X-100 (KPBS-BT). They were then pretreated in 1% H202 in KPBS for 20 rnin to reduce endogenous peroxidase and followed by incubation in a 10% swine serum solution in KPBS containing 0.3% Triton X-100 and 1% BSA for 20 min. The sections were then incubated for 2 days at 4°C in the specific antisera against NPY and CPON diluted 1:lOOO in KPBS containing 0.3% Triton X-100 and 1% BSA. After the incubation, the sections were washed for 3 X 10 rnin in KPBS-BT and then incubated with biotinylated swine anti- rabbit IgG (code #E353 , Dakopatts, Copenhagen) diluated 1:lOOO in KPBS-BT for 1 hr at room temperature and washed in KPBS-BT for 3 X 10 min. They were then incubated with the ABC streptavidin-horseradish peroxidase complex (code

1 Fig. 1. Camera lucida drawing of the sagittal section of the bovine epithalamic area showing the distribution of NPY- immunoreactive nerve fibers in the pineal gland and the adjacent epithalamic area. Ha = habenula, PC = posterior commissure, PG = pineal gland, pr = pineal recess.

#K377, Dakopatts, Copenhagen) diluted 1 :250 in KPBS-BT for 1 hr. The sections were then rinsed in KPBS-BT, KPBS, and 0.05 M Tris-Hcl buffer (pH 7.6) each for 10 min. Finally, they were reacted for peroxidase activity in a solution containing 0.025% DAB and 0.01% H20, in 0.05 M Tris-Hcl buffer (pH 7.6) for 10 to 20 mins. After rinsing in distilled water 2 X 5 min, the sections were placed on gelatinized glass slides, dried and coverslipped with DepexB.

Absorption controls

Sections were incubated with sera that had been absorbed with synthetic NPY and CPON (50 pg peptideiml diluted antiserum).

Results

The bovine pineal gland is cone-shaped and extends from the habenular commissure rostrally to the posterior commissure caudally. Its base is in contact with the third ventricle from where the gland extends in superior-caudal direction to be located on the pretectal area with its tip between the superior colliculi (Fig. 1). The gland is covered by a fairly thick pial capsule from where septae penetrate into

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Neuropeptide Y in the bovine pineal

are outside of the gland. The presence of a large number of NPY-containing fibers in the pial capsule of the gland indicates an origin of these nerve fibers from the superior cervical ganglion as shown in other species [Zhang et al., 1991; Li and Welsh, 19911. NPY classically colocalizes with noradren- aline in the sympathetic nervous system [Uddman et al., 19801. In the pineal of the rat [Reuss and Moore, 1989; Zhang et al., 19911, Mongolian gerbil (Shiotani et al., 1986), and sheep [Cozzi et al., 19921, a high number of NPYergic fibers originate from perikarya in this ganglion. However, it is interesting that few NPY-IR fibers in the rostra1 part of the rat pineal complex [Zhang et al., 19911 and many NPY-IR nerve fibers in the sheep pineal gland [Cozzi et al., 19921 remain after bilateral removal of the superior cervical ganglion.

In this study several NPY-containing nerve fibers were present in pineal stalk and in the habenular and posterior commissural areas. The possiblity exists that these fibers originate in the superior cervical ganglia and enter the pineal gland to continue into the brain via the pineal stalk as shown for the sympathetic nerve fibers in the rat (Bjorklund et al. , 1972). On the other hand, the NPY fibers might also originate from neurons in the central nervous sys- tem. In this study, the presence of a central inner- vation of the bovine pineal gland was supported by the presence of NPY-IR nerve fibers in the stalk dichotomizing in direction of the pineal. Recent studies in mink, rat, and sheep [Mdler et al., 1990; Zhang et al. 1991 ; Cozzi et al., 19921 have shown that the NPYergic innervation of the mammalian pineal does not exclusively originate from the peripheral sympathetic nervous system. However, the origin of the central NPY-containing fibers of bovine have not been elucidated in this study. Recently, a direct projection from the intergenicu- late leaflet to the pineal gland have been shown in the rat [Mikkelsen and Meller, 19901 and gerbil [Mikkelsen et al., 19911. This leaflet contains NPY-positive perikarya and is known to project to the suprachiasmatic nucleus [Card and Moore, 19891.

In this study, some NPY-containing fibers were observed in the posterior commisure. The origin of these fibers have to be elucidated. However, a possibility exists that they might originate from perikarya in the pretectal area, which in several species contains NPYergic perikarya [Chronwall et al., 19851.

Functionally, the anatomical location of NPY- containing nerve fibers in the bovine pineal gland indicates an influence on both the pineal vascular system as well as the pinealocyte itself. An influ-

the gland separating the parenchyma into smaller lobules.

lmmunohistochemical localization of NPY/CPON

By use of the antibody against CPON and also the antibody against NPY, a dense network of immu- noreactive nerve fibers were observed throughout the bovine pineal gland. No apparent difference in the density or in the location of the immunoreactive nerve fibers was observed when the two antibodies were used for staining. A large number of NPY/ CPON-immunoreactive (IR) nerve fibers were ob- served throughout the gland (Figs. 1 , 2). At the periphery, bundles of immunopositive fibers from the subarachnoid space entered the gland by pierc- ing the pial capsule (Fig. 3). Within the gland, the IR nerve fibers were located in the perivascular spaces but penetrated intraparenchymally , and were here located as a diffuse network between the cluster of pinealocytes (Fig. 4).

The IR nerve fibers exhibited various shapes and sizes. The intraparenchymal fibers usually were thin branching fibers (diameter 0.2-0.5 pm), often endowed with bouton-like varicosities (Fig. 4). Some thin fibers were occasionally endowed with large varicosities (diameter 3-6 p.m) (Fig. 5 ) . Finally, some fibers, especially located in the peripheral and basal part of the gland close to the stalk, were thick (diameter 1-1.5 pm) and endowed with only few boutons en passage.

IR fibers were also detected in the stria medul- lark thalami, the habenular and posterior commis- sures (Fig. 1). Several of these IR nerve fibers in the habenular and posterior commissures could be fol- lowed to the pineal stalk and further into the gland itself. Nerve fibers in the pineal stalk were often observed to dichotomize in directions of the pineal gland.

Controls

No staining was observed after absorption of the diluted antisera with the specific peptide.

Discussion

This study demonstrates the presence of an intense innervation of the bovine pineal gland with NPY - CPON-IR nerve fibers. The density of NPY-con- taining fibers observed in this species is as high as previously shown in the rat [Zhang et al., 19911 and sheep pineal gland [Cozzi et al., 19921.

The lack of immunoreactive cell bodies in bovine pineals shows that the source(s) of the NPY fibers

55

Phansuwan-Pujito et a].

Fig. 2 . A dense network of NPY-IR nerve fibers is seen both perivascularly and intraparenchymally in the bovine pineal gland. Antibody #8999. X366. Fig. 3. NPY-IR nerve fibers (arrows) in the pineal capsule and in the outer part of the gland. Some fibers enter the gland from the capsule. SS = subarachnoidal space. Antibody #8999. x590. Fig. 4. A diffuse network of branching NPY-IR fibers in the parenchyma of the gland. Antibody #8999. X590. Fig. 5 . A bundle of medium sized NPY-IR nerve fibers endowed with large boutons (bent arrow) en passage. Antibody #8999. X590.

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Neuropeptide Y in the bovine pineal

ence of NPY on the vascular system has been demonstrated in several studies [Edvinsson et al., 19831. Endogenous NPY is able to induce vason- constriction [Potter, 19881.

With regard to an influence on the pinealocyte, conflicting reports have been presented. Thus, in- travascular injection of NPY in the rat has been reported to stimulate the pinealocyte indole metab- olism by increasing the activity of serotonin N-acetyltransferase during the day but to inhibit the enzyme activity at night [Reuss and Schroder, 19871. Further, Vascas et al. [1987], have reported that NPY significantly enhanced the basal and noradrenaline-induced stimulation of melatonin production in rat. In cultures of the ovine pinealo- cytes, NPY had neither direct effect on the indole metabolism [Morgan et al., 19881 nor interaction with noradrenergic stimulation of cAMP production or melatonin synthesis [Williams et al., 19891. Recently, however, NPY was reported to inhibit a noradrenaline stimulated rise in cAMP in cultures rat pinealocytes [Olcese, 19911.

Summarizing, our immunohistochemical study of bovine pineal glands demonstrates a dense perivascular and intraparenchymal network of NPY/ CPON-IR nerve fibers. These fibers could be of dual origin, from perikarya in the brain or from the superior cervical ganglion. The origin of most of these fibers is probably the superior cervical gan- glion, but the morphology of the immunoreactive nerve fibers in the pineal stalk indicates that some of these fibers might originate from the brain and enter the pineal gland via the stalk.

Literature Cited

ALLEN, J.S., T.E. ADRIAN, J.M. ALLEN, K. TATEMOTO, T.J. CROW, S.R. BLOOM, J.M. POLAK (1983) Neuropeptide Y distribution in the rat brain. Science 221 :877-879.

BJORKLUND, A, , CH.H. OWMAN, K.A. WEST (1972) Peripheral sympathetic innervation and serotonin cells in the habenular region of the rat brain. Z. Zellforsch. 127576579.

CARD, J.P., R.Y. MOORE (1989) Organization of the lateral geniculate hypothalamic connections in the rat. J. Comp. Neurol. 284: 135-147.

CHRONWALL, B.M., D.A. DIMAGGIO, V.J. MASSARI, V.M. PICKL, D.A. RUGGIERO, T.L. O'DONOHEU (1985) The anat- omy of neuropeptide Y-containing neurons in the rat brain. Neuroscience 15:1159-1181.

COZZI, B., J.D. MIKKELSEN, J-P. RAVAULT, M. M@LLER (1992) Neuropeptide Y (NPY) and C-flanking NPY in the pineal gland of normal and superior ganglionectomized sheep. J. Comp. Neurol. 316:238-250.

EBADI, M . , T.D. HEXUM, R.F. PFEIFFER, P. GOVITRAP~NG (1989) Pineal and retinal peptides and their receptors. Pineal Res. Rev. 7:l-156.

EDVINSSON, L., P. EMSON, J. MCCULLOCH, K. TATEMOTO, R. UDDMAN (1983) Neuropeptide Y: Cerebrovascular innerva- tion and vasomotor effects in the cat. Neurosci. Lett. 43:79- 84.

GOLDMAN, B.D., J.M. DARROW (1983) The pineal gland and mammalian photoperiodism. Neuroendocrinology 37:38& 396.

KORF, H.W., M. M@LLER (1984) The innervation of the mammalian pineal gland with special reference to central pinealopetal projections. Pineal Res. Rev. 2:41-86.

LI, K., M.G. WELSH (1991) Tyrosine hydroxylase and neu- ropeptide-Y immunoreactivity in pineal glands developing in situ and in pineal grafts. Cell Tissue Res. 264:515-527.

LUNDBERG, J.M., L. TERENIUS, T. HOKFELT, C.R. MARTLING, K. TATEMOTO, V. MUTT, J. POLAK, S . BLOOM, M. GOLD- STEIN (1982) Neuropeptide Y-like immunoreactivity in pe- ripheral noradrenergic neurons and effects of NPY on sym- pathetic function. Acta Physiol. Scand. 1 16:477-480.

MIKKELSEN, J.D., B. COZZI, M. M@LLER (1991) Efferent projection from lateral geniculate nucleus to the pineal com- plex of the Mongolian gerbil (Meriones unguiculatus). Cell Tissue Res. 264:95-102.

MIKKELSEN, J.D., M. M ~ L L E R (1990) A direct neural connec- tion from the intergeniculate leaflet of the lateral geniculate nucleus to the deep pineal gland of the rat demonstrated with Phaseolous vulgaris leucoagglutinin. Brain Res. 520:342- 346.

M@LLER, M., J.D. MIKKELSEN, L. MARTINET (1990) Innerva- tion of the mink pineal with neuropeptide Y (NPY)-containing nerve fibers. An experimental immunohistochemical study. Cell Tissue Res. 261:477483.

MOLLER, M., J.-P. RAVAULT, B. Cozzi, E.-T. ZHANG, P. PHANSUWAN-PUJITO, P.J. LARSEN, J.D. MIKKELSEN (1991) The multineuronal input to the mammalian pineal gland. In: Advances in Pineal Research. Vol. 6. A. Foldes, R.J. Reiter, eds. John Libbey, London, pp. 3-12.

MORGAN, P.J., L.M. WILLIAMS, W. LAWSON, G. RIDDOCH (1988) Stimulation of melatonin synthesis in ovine pineals in vitro. J. Neurochem. 50:75-81.

OLCESE, J. Y. NEUROPEKIDE: (1991) An endogenous inhibitor of norepinephrine-stimulated melatonin secretion in the rat pineal gland. J. Neurochem. 57:943-947.

PEVET, P., M. MASSON-PEVET, B. VIVIEN-ROELS, H. BONN (1986) Photoperiod, temperature, melatonin, 5-methox- ytryptamine and seasonal reproduction: Some data on the golden hamster. In: Advances in Pineal Research, Vol. 1. R. Reiter, M. Karasek, eds. John Libbey, London, pp. 185-196.

POTTER, E.K. (1988) Neuropeptide Y as an autonomic neuro- transmitter. Pharmacol. Ther. 37:25 1-273.

REUSS, S . , R.Y. MOORE (1989) Neuropeptide Y-containing neurons in the rat superior cervical ganglion: Projections to the pineal gland. J. Pineal Res. 6:307-316.

REUSS, S . , H. SCHRODER (1987) Neuropeptide Y effects on pineal melatonin synthesis in the rat. Neurosci. Lett. 74:158- 162.

SCHON, F., J .M. ALLEN, J.C. YEATS, Y. ALLEN, J. BALLESTA, J. POLAK, J.S. KELLY, S.R. BLOOM (1985) Neuropeptide Y innervation of the rodent pineal gland and cerebral vessels. Neurosci. Lett. 57:65-71.

SCHRODER, H. (1986) Neuropeptide Y (NPY)-like immunore- activity in peripheral and central nerve fibers of the golden hamster (Mesocricetus aurutus) with special respect to pineal gland innervation. Histochemistry 85:32 1-325.

SCHRODER, H . , L. VOLLRATH (1986) Neuropeptide Y (NPY)- like immunoreactivity in the guinea-pig pineal organ. Neuro- sci. Lett. 63:285-289.

SHIOTANI, Y., M. YAMANO, S. SHIOSAKA, P.C. EMSON (1986) Distribution and origins of substance P (SP)-, calcitonin gene-related peptide (CGRP)-, vasoactive intestinal polypep- tide (VIP)- and neuropeptide Y (NPY)-containing nerve fibers in the pineal gland of gerbils. Neurosci. Lett. 70:187-192.

57

Phansuwan-Pujito et al.

SUGDEN, D. (1991) Adrenergic mechanisms regulating pineal melatonin synthesis. In: Advances in Pineal Research, Vol. 5 . J. Arendt, P. Ptvet, eds. John Libbey, London, pp. 33-38.

TATEMOTO, K. M. CALQUIST, V. MUIT (1982) Neuropeptide Y: A novel brain peptide with structural similarities to peptide YY and pancreatic peptide. Nature 0268:659460.

UDDMAN, R., J. ALUMETS, R. HAKANSON, J. LOREN, F. SUNDLER (1980) Vasoactive intestinal peptide (VIP) occurs in the nerves of the pineal gland. Experientia 36: 11 19-1 120.

VACAS, M.J., S.M.J. KELLER, E.N. PEREYRA, G.S. ETCHE- GOYEN, D.P. CARDINALI (1987) In vitro effect of neuropeptide

Y on melatonin and norepinephrine release in rat pineal gland. Cell Mol. Neurobiol. 7:309-3 15.

WILLIAMS, L.M., P.J. MORGAN, G. PELLETIER, G.I. RIDWCH, W. LAWSON, G.R. DAVIDSON (1989) Neuropeptide Y (MY) innervation of the ovine pineal gland. J. Pineal Res. 7:345- 353.

ZHANG, E-T., J.D. MIKKELSEN, M. M ~ L L E R (1991) Tyrosine hydroxylase- and neuropeptide Y-immunoreactive nerve fi- bers in the pineal complex of untreated rats and rats following removal of the superior cervical ganglion. Cell Tissue Res. 265163-71.

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