Neuropeptide Y in the mammalian pineal gland

Neuropeptide Y in the Mammalian Pineal GlandJENS D. MIKKELSEN* AND MORTEN MØLLERDepartment of Anatomy B, University of Copenhagen, Copenhagen, Denmark

KEY WORDS neuropeptide Y (NPY); ProNPY; pineal gland; sympathetic innervation; norepi-nephrine; noradrenaline; peptide processing; colocalization; immunohistochemis-try; electron microscopy; rat; mammal

ABSTRACT The present review describes the anatomy of the neuropeptide (NPY)ergic innerva-tion of the mammalian pineal gland with emphasis on the rat. The proNPY-molecule is post-translationally processed by a single cleavage to neuropeptide Y (NPY) and a C-terminal peptide ofNPY (CPON). NPY is C-terminally amidated, and the amidation is essential for binding of NPY toits corresponding receptor(s). Since no proNPY has been detected in rat pineal extracts, it isconsidered that proNPY is immediately processed to its final products in the gland. In the rat,numerous NPY- and CPON-immunoreactive (ir) nerve fibers are present in the capsule of thesuperficial pineal gland and in the pineal parenchyma, mostly related to the connective tissue spacesand the vasculature of the gland, but also present between the pinealocytes. Furthermore, asubstantial number of fibers was observed in the deep pineal gland, the pineal stalk, and theunderlying epithalamus. Occasionally, NPY- or CPON-immunoreactive fibers were found adjacentto the stria medullaris and in the posterior commissure, which could be followed to the adjacent deeppineal gland. At the ultrastructural level, the NPY-immunoreactivity was confined in boutonscontaining large granular vesicles (100–200 nm) as well as small (40–60 nm) granular vesicles.Some terminals were located in very close apposition to the pinealocyte cell membrane. Terminalswere identified in perivascular spaces, but synaptic contacts between the immunoreactive terminalsand pinealocytes were never observed. These data show that NPY is highly concentrated in nervefibers throughout the rat pineal complex. Double-fluorescence histochemistry using tyrosinehydroxylase as marker for catecholaminergic fibers and NPY revealed that nearly all NPYergicfibers co-stored tyrosine hydroxylase in the superficial pineal gland. A minor portion of bothimmunoreactivities was not colocalized. In accordance, about 65% of the neurons in the superiorcervical ganglion contained both CPON and tyrosine hydroxylase. In bilateral superior cervicalganglionectomized rats, a few NPY-ir nerve fibers remained mostly in the pineal capsule, but fewfibers were also found in the superficial pineal parenchyma. Contrarily, only a moderate decreasewas observed in the number of immunoreactive fibers in the deep pineal gland, and no reduction wasobserved in the adjacent epithalamus. In the ganglionectomised rats, co-localisation of tyrosinehydroxylase and NPY in intrapineal nerve fibers was not observed either in the superficial pinealgland, nor in the deep pineal gland. These results together with the available literature show thatNPY is a sympathetic transmitter, and its actions in the pineal gland are, therefore, associated withthe well-documented roles of noradrenaline. Possible roles of NPY in pineal biochemistry andphysiology are discussed. Microsc. Res. Tech. 46:239–256, 1999. r 1999 Wiley-Liss, Inc.

INTRODUCTIONThe pineal gland secretes melatonin in a characteris-

tic circadian rhythm and forms an essential organ inthe photoneuroendocrine system allowing mammalianspecies to keep time. In certain mammalian species, thepineal gland is also of crucial importance in the controlof reproduction (Reiter, 1980, 1991). The mammalianpineal gland is densely innervated by sympatheticfibers originating from the superior cervical ganglia(Moore and Klein, 1974). The noradrenergic inputstimulates the synthesis of melatonin and thereby thefunctional state of the pineal gland. Release of nor-adrenaline from the sympathetic nerve terminals isconsidered the only signal conveying the circadian

information from the brain to the pineal gland (Mooreand Klein, 1974).

Neuropeptide Y (NPY) is one of several neuropep-tides that have been identified in the mammalianpineal gland (Cozzi et al., 1992; Mikkelsen et al., 1999;Møller et al., 1990, 1994; Reuss and Moore, 1989; Schonet al., 1985; Shiotani et al., 1986; Zhang et al., 1991).

Contract grant sponsor: Ivan Nielsen Foundation; Contract grant sponsor:NOVO Nordisk Foundation; Contract grant sponsor: Danish Medical ResearchCouncil to the Center for Cellular Communication.

*Correspondence to: Jens D. Mikkelsen, Department of Molecular Cell Biology,Zealand Pharmaceuticals A/S, Smedeland 26 B, 2600 Glostrup, Denmark.E-mail: [email protected]

Accepted 24 May 1999

MICROSCOPY RESEARCH AND TECHNIQUE 46:239–256 (1999)

r 1999 WILEY-LISS, INC.

Several studies have shown that NPY is present in thesympathetic nervous system, where it occurs togetherwith noradrenaline in the postganglionic fibers (Lund-berg et al., 1982). Also, in the rat pineal gland themajority, if not all, of the NPY-containing nerve fibersare postganglionic sympathetic noradrenergic nervesoriginating from the superior cervical ganglia (Reussand Moore, 1989; Schon et al., 1985; Zhang et al., 1991).

The functional significance of the noradrenergic in-put to the pineal gland is well known (Korf et al., 1998).Noradrenaline stimulates the synthesis and release ofmelatonin via postsynaptic b1-receptors (Deguchi andAxelrod, 1972). In addition, noradrenalin stimulatesproduction of intracellular cAMP, protein kinase Aactivation, phosphorylation of the cyclic AMP responseelement binding protein CREB, transcription of theinducible cyclic AMP element repressor (ICER) andserotonin-N-acetyl transferase (NAT) within the pine-alocytes (Baler et al., 1997; Chik et al., 1997; Coon etal., 1997; Stehle et al., 1993). In contrast, the role of theco-stored peptide, NPY, is still unclear. No effect of NPYon the release of melatonin was found in a primaryovine or rat pineal organ cultures (Morgan et al.,1988a,b; Olcese, 1991; Simonneaux et al., 1994). In ratpineal cultures, NPY inhibits the stimulatory effect ofnoradrenaline on melatonin release (Olcese, 1991; Si-monneaux et al., 1994). The effect of NPY on thenoradrenergic transmission is probably transmitted viainhibitory G-proteins in the membrane reducing theactivity of adenylate cyclase in the target cells (Fred-holm et al., 1985; Kassis et al., 1987). In accordance,NPY Y1 receptor inhibits adenylate cyclase activity in anumber of cell lines studied, and it is likely that thesame effect occurs in the pinealocyte, because the samereceptor is found in the pineal gland (Mikkelsen et al.,1999; Olcese, 1991; Simonneaux et al., 1994). Takentogether, NPY is present in high concentrations in therat pineal gland, but its direct effect on the pinealocyte,at least in regulating melatonin synthesis and secre-tion, needs further clarification.

In order to understand the physiology of NPY in therat pineal gland in more detail, a careful analysis of theneuroanatomy of the NPYergic system in mammals isrequired. Earlier studies have shown the presence ofNPYergic innervation of the rat pineal gland at thelight microscopical level (Mikkelsen et al., 1999; Reussand Moore, 1989; Schon et al., 1985; Zhang et al., 1991).With light microscopy, the relation between the NPYer-gic afferents and the pinealocytes or the vasculaturecannot be resolved. Therefore, the distribution of NPY

in the superficial pineal was studied at the ultrastruc-tural level as well.

The present study aims to analyze in detail theNPYergic afferent nerves in the rat pineal gland, andcompare this innervation with the NPYergic innerva-tion with other species reported in the literature. Thismay be important because the role of the pineal andmelatonin shows major differences among mammalianspecies (Reiter, 1980). The distribution and content ofNPY in the pineal gland also vary considerably betweenmammalian species (Cozzi et al., 1992; Mikkelsen et al.,1999; Mikkelsen and Mick, 1992; Møller et al., 1990,1994; Zhang et al., 1991).

Another aim of the study was to determine the originof the NPYergic nerve fibers. In rat and gerbil, NPY-irnerve fibers are known to originate from the superiorcervical ganglia (Reuss and Moore, 1989; Shiotani etal., 1986; Zhang et al., 1991), but this may not be theonly source (Zhang et al., 1991). It was further ana-lysed, by using double immunofluorescence, how manyof the sympathetic neurons in the superior cervicalganglia also contain NPY. The present study alsoexpands previous observations by quantitative estima-tions of the origin of the NPYergic nerve fibers fromsympathetic and non-sympathetic sources.

MATERIALS AND METHODSAntisera and Peptides

Rabbit antisera against NPY used in the presentinvestigation (nos. 8182 and 337) have been previouslycharacterized (Mikkelsen et al., 1993; Mikkelsen andO’Hare, 1991; O’Hare and Schwartz, 1989). Antiserumno. 337 recognizes an unknown epitope in the middle ofthe molecule, whereas no. 8182 recognizes both theamidated tyrosine and another epitope of the peptide.The third antiserum raised against Cys-NPY32-36-amide (no. 8999) has only high affinity for NPY in itsamidated form, but not for desamidoNPY. Antiseraagainst CPON were purchased from Cambridge Re-search Biochemical (Cheshire, UK) or Nova-biochem(Laufelfingen, Switzerland). Antisera against tyrosinehydroxylase and dopamine b-hydroxylase were pur-chased from Immunonuclear (Stillwater, IL) and Eu-gene Technology (Allendale, NJ), respectively.

Tissues and FixationA total number of 40 adult Wistar rats housed under

a photoperiod of 12h light/12h darkness (lights on0600h) with free access to food and water were used inthese studies. All animals were obtained from breedingfacilities at the Panum Institute, Copenhagen. Prin-

Abbreviations

ChP choroid plexusCPON C-terminal flanking peptide of NPYDBH dopamine b-hydroxylasedp deep pineal glandhc habenular commissureNPY neuropeptide Ypc posterior commissurepr pineal recessPYY peptide YYSCO subcommissural organTH tyrosine hydroxylaseVIP vasoactive intestinal peptide

Figs. 1–5. Distribution of NPY-ir nerve fibers in the superficialpineal gland, the pineal stalk, and the choroid plexus. In the dorsalpart of the superficial pineal gland, the pineal capsule containsnumerous NPY-ir nerve fibers (thick arrows; Fig. 1), some of whichpenetrate into the pineal parenchyma along the perivascular spaces(open arrows, Fig. 1). In the pineal gland NPY-ir nerve fibers can befollowed from the dorsal surface into the perivascular spaces (arrows;Fig. 2). The choroid plexus located dorsal to the deep pineal glandcontains several NPY-ir nerve fibers. Note also the smooth fibers in thepineal stalk (straight arrows; Fig. 3). The fibers in the choroid plexusare both smooth and thick (large arrows), or thinner with varicosities(small arrows; Fig. 4). In another part of the choroid plexus, onlydelicate varicose nerve fibers are observed (Fig. 5). dp 5 deep pinealgland; hc 5 habenular commissure; Stalk 5 pineal stalk. Scale bars(Figs. 1, 2, 4, and 5) 5 25 µm; (Fig. 3) 5 100 µm.

240 J.D. MIKKELSEN AND M. MØLLER

Figs. 1–5.

Figs. 6–9. The figures illustrate the distribution of NPY-ir nervefibers in the rat superficial pineal gland. Low-power microphotographdemonstrates a dense homogenous distribution of NPYergic nervefibers in the gland (Fig. 6). At higher power, a bundle of smooth nervefibers are seen (large arrows; Fig. 7). In the peripheral part of the

superficial pineal gland, the NPY-ir nerve fibers are located either inperivascular spaces indicated by open arrows (Figs. 8 and 9) orsmaller single nerve fibers with boutons en passage in the parenchyma(small arrows; Fig. 8). Scale bars (Fig. 6) 5 100 µm; (Figs. 7–9) 550 µm.


ciples of laboratory animal care and specific nationallaws were followed. Bilateral removal of the superiorcervical ganglia was performed in 10 rats during thedaytime. The operated animals were sacrificed after 2,3, 6, and 8 weeks of survival.

The animals were anaestetized with tribromethanol(250 mg/kg) i.p. during the day and perfused with 50mM phosphate-buffered saline (PBS, pH 7.4) with15,000 IU/l heparin for 3 minutes followed by perfusionfor 15 minutes with 400 ml 4% paraformaldehyde in0.1M phosphate buffer (pH 7.4). For ultrastructuralstudies, the fixative also contained 0.1% glutaralde-

hyde (MERCK, Darmstadt, Germany). The brains wereremoved and immersed in the same fixative for anadditional 16 hours at 4°C. For ultrastructural studies,the epithalamic region was removed and 50–100-µm-thick sections were cut in a vibratome and stored inPBS. For light microscopical studies, the tissue wasthen infiltrated with a 30% sucrose-PBS solution for 3days and 40-µm-thick serial cryostat sections were cutand placed in PBS to which 0.02% KCl was added(KPBS) at 4°C until further use. Other brains were cutsagittally into 20-µm serial sections, and placed ongelatinized glass slides.

Figs. 10–12. Illustrations of the distribution of CPON-ir nervefibers in the deep pineal gland. The CPON-ir nerve fibers are presentin the deep pineal gland (dp), whereas a lower number are observed inthe posterior (pc) and habenular (hc) commissures (Fig. 10). A singlefiber is running through the posterior commissure towards the deeppineal gland (open arrow, Fig. 11). Other fibers are present close to the

pineal recess (small arrow, Fig. 11). A micrograph is showing the denseinnervation of CPON-ir nerves in the deep pineal gland. Note that thesingle fibers are of different morphologies, and that some of the fibersare related to the space of the pineal recess (pr) (Fig. 12). ChP 5Choroid plexus. Scale bars (Fig. 10) 5 100 µm; (Figs. 11, 12) 5 50 µm.

243NPY IN THE MAMMALIAN PINEAL GLAND

ImmunohistochemistryThe sections were processed for immunohistochemis-

try by the use of the streptavidin enzyme histochemicaltechnique (Hsu et al., 1981). The sections were rinsedtwice for 5 minutes in KPBS (pH 7.4) and pretreated in1% H2O2 in KPBS for 10 minutes and then incubatedfor 20 minutes in a 4% swine serum solution in KPBScontaining 0.3% Triton X-100 and 1% BSA. The sectionswere then incubated in the primary antiserum for 24hours at 4°C. The dilutions were as follows: NPY(antisera nos. 337 and 8182), 1:1,000; Cys-NPY (32–36)amide (antiserum no. 8999), 1:400; CPON, 1:2,000;tyrosine hydroxylase, 1:4,000 and dopamine b-hydroxy-lase, 1:40. Usually the concentrations of the primaryantisera were increased 2-fold, if the sections werereacted on slides. The sections were then washed inKPBS to which 0.25% bovine serum albumin (BSA) and0.1% Triton X-100 were added (KPBS-BT) for 3 3 10minutes followed by incubation with a biotinylatedswine anti-rabbit IgG (no. E353, DAKO, Copenhagen,Denmark) diluted 1:400 in KPBS-BT for 60 minutes atroom temperature. When the sections were reacted fortyrosine hydroxylase-immunoreactivity, a biotinylatedrabbit anti-mouse (no. E354, DAKO) diluted 1:400 wasused, because the primary antiserum was raised inmouse. They were next washed for 3 3 10 minutes inKPBS-BT, and finally incubated for 60 minutes at roomtemperature in anABC-streptavidin-horseradish peroxi-dase complex (code no. K377, DAKO) diluted 1:1,000 inKPBS-BT. After washing in KPBS-BT for 10 minutes,in KPBS alone for 10 minutes and in 50 mM Tris/HClbuffer (pH 7.6) for 10 minutes, the sections were reactedfor peroxidase activity by incubation with a solution of1.25 mg/l diaminobenzidine (DAB) in 0.05 M Tris/HCl-buffer (pH 7.6) and 0.03% H2O2 for 20 minutes. Afterwashing for 2 3 5 minutes in distilled water, thesections were mounted on gelatinized glass slides,dried, dehydrated in a series of ethanols, and embeddedin Depext. Some adjacent sections were counterstainedin thionine after the DAB-reaction.

Double ImmunohistochemistrySections were prepared as described above and incu-

bated in a mixture of a polyclonal rabbit CPON antise-rum and a mouse monoclonal TH-antiserum. The sec-tions were washed and incubated in a mixture ofFITC-conjugated swine anti rabbit (1:10) and biotinyl-ated swine anti-mouse (E354). After a careful wash, thesections were incubated in streptavidin-Texas Red,washed, dried, and embedded in glycerol.

Quantification of TH- and NPYergic Nerve FibersThe density of fibers in the pineal gland was counted

in a bright-field microscope within a standarised area.The number of single- and dual-labeled perikarya inthe SCG was counted in the fluorescent microscope.

Electron MicroscopyThe vibratome sections were then incubated in a

rabbit anti-NPY (no. 8182) diluted 1:1.000 in PBS with0.25% bovine serum albumin (BSA) for 2 days at 4°C.After 3 3 15 minutes rinse in PBS, the sections wereincubated in biotinylated swine anti-rabbit IgG (no.353, DAKO) diluted 1:400 in PBS with 0.25% BSA for 2

hours at room temperature. After 3 3 15 minutes rinsein PBS, the sections were incubated in ABC-streptavi-din-horseradish peroxidase complex (no. K377, DAKO)diluted 1:1,000 in PBS with 0.25% BSA for 2 hours.After 3 3 15 minutes wash in PBS the sections werepostfixed in 4% glutaraldehyde in 0.1 M phosphatebuffer for 2 hours. Finally, after 3 3 15 minutes wash inTris/HCl buffer, the sections were reacted in diaminoben-zidine. After washing 3 3 15 minutes in phosphatebuffer, the sections were osmificated in 2% OsO4 in 0.1M phosphate buffer for 2 hours, the block was stained in1% uranyl acetate in water, dehydrated in increasingconcentrations of ethanol, and flat embedded via propyl-ene oxide in Epont. After polymerization, the sectionswere polymerized to a prepolymerized Epon block andthin sections, with a gray to silver interference color,were cut and counterstained with lead citrate. Thesections were viewed and photographed in a Philips 400electron microscope operated at 60 or 80 kV.

RESULTSDistribution of NPY/CPON-Immunoreactive (ir)

Nerve Fibers in the Pineal ComplexGel chromatography has shown that antisera di-

rected against desamidoNPY and Cys-NPY32-36amidein the pineal gland recognize the same molecule, namelyNPY(1-36)amide (Mikkelsen et al., 1999). Therefore,nerve fibers exhibiting immunoreactivity for either ofthese antisera will be referred to as NPY-ir nerve fibers.

The distribution of NPY/CPON-ir nerve fibers wasexamined in the rat pineal complex and in the epithala-mus. NPY/CPON-ir nerve fibers were found throughoutthe rat pineal complex and it is very likely that CPONand NPY exhibit 100% co-localization in these nerves.We, therefore, refer to the fibers as NPY/CPON-irnerves unless otherwise noted.

NPY/CPON-ir nerve fibers were found in the menin-geal tissue of the pineal capsule, in fibers surroundingpial arteries, and in the choroid plexus adjacent to thedeep pineal gland (Figs. 1–5). The NPY/CPON-ir nervefibers in these structures were numerous and displayeddifferent morphologies. Bundles of nerve fibers wereobserved in the choroid plexus (Figs. 4, 5) and in themeningeal tissue covering the superficial pineal gland(Fig. 1). The bundles of NPY/CPON-ir nerve fibersoriginating from the superior cervical ganglia (seebelow) reached the pineal at the caudal tip of thesuperficial pineal gland, from where thick non-varicosefibers entered the pineal capsule (Figs. 1 and 2). Fromthe capsule, these fibers penetrated into the gland andwere followed into large and small interlobular connec-tive tissue spaces close to the intrapineal blood vessels(Figs. 2, 6, 7, and 9). Single fibers were intermingledbetween groups of pinealocytes (Figs. 1, 6, 8, and 9).

The rostral part of the pineal complex, comprisingthe body of the deep pineal gland together with thepinealocytes attached to the pineal stalk, and theepithalamic area comprising the habenular commis-sure, and the dorsal part of the posterior commissure,were examined for NPY/CPON-ir. A fairly high numberof NPY/CPON-ir fibers were observed in the deeppineal gland (Figs. 10–13). The fibers were relativelythick exhibiting large dense immunoreactive nerveendings (about 2 µm in size), associated with theendocrine tissue (Figs. 10–12). Other nerves were pres-


Figs. 13–16. Photomicrographs of median sections of the deeppineal complex, the pineal stalk, and the proximal part of the superiorpineal gland illustrating the distribution of CPON-ir nerve fibers. Asection of the pineal deep pineal gland and the proximal part of thepineal stalk with varicose and smooth fibers (small arrows) (Fig. 13).In the mid-portion of the pineal stalk, CPON-ir nerve fibers are

present (small arrows) (Fig. 14). In the distal part of the pineal stalksingle fibers are present, and in some cases an accumulation ofCPON-ir fibers is present in the extreme proximal part of thesuperficial pineal gland (open arrows, Fig. 15). Smooth and varicosefibers are present in the stalk in Figure 16. ChP 5 choroid plexus.Scale bars (Figs. 13, 15–16) 5 100 µm; (Fig. 16) 5 50 µm.


ent in the ependyma of which some terminate close tothe ventricular lumen (Figs. 11, 12). In the posteriorand habenular commissures, the number of immunore-active fibers was low. Occasionally, a single fiber wasseen either passing along the posterior commissure orpenetrating the posterior commissure in direction ofthe deep pineal gland (Fig. 11). A few NPY/CPON-irfibers were observed in the stria medullaris projections,whereas a dense accumulation of positive nerve fiberswas found laterally to the subcommissural organ (Fig.10). Thick NPY/CPON-ir nerve fibers and thinner onesendowed with boutons of variable sizes were observedin the pineal stalk (Figs. 3, 14, 15, 16). In mediansections, the NPY/CPON-ir nerves could be followed fornearly the entire extension of the stalk (Figs. 3, 14). Noimmunoreactive cell bodies were observed in the ratpineal complex.

Comparing Staining With Various AntiseraFibers immunoreactive for all antigens were distrib-

uted throughout the pineal parenchyma, and wereapparently homogenously distributed in the organ. Thefibers displayed the same morphology and distributionindependent of the antiserum used. By comparing thedistribution of the immunoreactive fibers to which theantisera were directed, the number of CPON- anddesamidoNPY-ir (using antiserum nos. 8182 or 337)

fibers was slightly higher than the number of NPYam-ide-ir (using antiserum no. 8999) fibers in any part ofthe pineal complex (Figs. 17–19).

Ultrastructural ObservationsBy use of the preembedding immunocytochemical

technique, the ultrastructure of the rat pineal glandwas satisfactory in most parts of the vibratome sec-tions. Many NPY-ir nerve terminals were observed (Fig.20) located throughout the pineal gland, both in theperivascular spaces (Figs. 20 and 23) and in the paren-chyma between the pinealocytes (Figs. 21 and 22). Mostof the positive terminals were identified as sympatheticnerve terminals due to the presence of small transmit-ter vesicles confining an electron dense core (Fig. 21).Some sympathetic nerve terminals were non-immuno-reactive (Figs. 20 and 23). The NPY-ir nerve terminals

Figs. 20–21. Electron micrograph showing many intrapineal NPY-irnerve terminal and some non-immunoreactive terminals (small bentarrows) (Fig. 20). Electron micrograph of an intracellular spacebetween four pinealocytes. A NPY-ir sympathetic nerve terminal(black arrow) is located very close to two pinealocytes without makingsynaptic contact with the pinealocytes (Pi). A non-immunoreactive,sympathetic nerve terminal (bent arrow) is seen close to the labeledone (Fig. 21). Scale bars (Fig. 19) 5 2 µm; (Fig. 20) 5 1 µm.

Figs. 17–19. The three figures illustrate the distribution of nervefibers immunoreactive for NPY-amide in the deep pineal gland (ar-rows; Fig. 17), and the superficial pineal gland (Figs. 18, 19). Fibersare seen both in the capsule (small arrows) and in the intralobular

spaces (open arrow; Fig. 18). The number of the fibers immunoreactivefor NPYamide is lower than the number of fibers visualized withantisera against NPY or CPON. Scale bars 5 100 µm.


Figs. 20 and 21.

were often located very close to the cell membrane ofthe pinealocyte (Fig. 21), but synapse-like contactswere never observed.

Distribution and Content of NPY-and CPON-Immunoreactivity

in Ganglionectomized RatsIn bilateral ganglionectomized animals, the number

of NPY/CPON-ir fibers was dramatically reduced in thesuperficial pineal compared to control animals. Inadjacent sections, the number of tyrosine hydroxylase-and dopamine b-hydroxylase-immunoreactive nerve fi-bers was also reduced drastically (not shown). However,the distribution of CPON/NPY-ir nerve fibers in thepineal parenchyma after superior cervical ganglionec-tomy was no longer homogenous. Whereas the superfi-cial pineal gland virtually lacked an innervation, thedeep pineal gland contained a slightly lower number offibers compared to normal and sham-operated animals.As illustrated in Figures 24–28, only a few immunoreac-tive fibers were identified in the capsule and the pinealparenchyma of the superficial pineal gland (Figs. 27,28). In contrast, in the deep pineal area, and the pinealstalk, the number of NPY/CPON-ir fibers was onlymoderately decreased (Figs. 24, 25). In the epithala-mus, and around the subcommissural organ, severalimmunoreactive fibers were observed with apparentlyno differences in density compared to normal animals.

NPY/CPON and Tyrosine Hydroxylase (TH)-irNeurons in the Pineal Gland and the Superior

Cervical GanglionIn the superficial pineal, double fluorescent staining

showed that most of the TH-ir nerve fibers containedboth TH and CPON (Figs. 29–32). However, few fiberscontained either TH or CPON. Animals sacrificed atvarious times after the ganglionectomises showed thesame loss of NPY-ir nerve fibers.

The relative density of TH and CPON-ir nerve fiberswas counted in 0.18 mm2 unit area. The density ofCPON-ir fibers was 123.9 6 6.0 and the density of TH-irfibers was 157.6 6 12.6 in this area of measurement(Fig. 33), showing that the NPYergic fibers were con-tained in almost the entire population of sympatheticnerve fibers.

In the superior cervical ganglion, 66% of the peri-karya were CPON-ir. Among the immunoreactive peri-karya, 57.9 6 1.3% contained both CPON and TH,13.7 6 2.5% contained CPON alone, and 28.4 6 2.8%contained only TH (Fig. 34). Though a quantitativeassessment of the absolute number of fibers containingboth TH and CPON was not carried out in the presentinvestigation, these estimations suggest that the rela-tive content of NPY in nerve fibers in the pineal gland ishigher than in other organs innervated by sympatheticnerves.

As mentioned above, the number of NPY-ir nervefibers in the ganglionectomised superficial pineal glandwas very low. However, double fluorescent stainingshowed that no fibers in the ganglionectomised ratco-stored TH and CPON (Figs. 35, 36).

DISCUSSIONThis study shows that both the superficial pineal

gland, the deep pineal gland, and the pineal stalk of the

rat are densely innervated by nerve fibers containingboth NPY(1-36)amide and CPON. Further, it was re-vealed that the majority of these fibers originatefrom the superior cervical ganglia but a minority offibers, especially located in the deep pineal and thepineal stalk originate from an unknown extrasympa-thetic source. The origin of the extrasympatheticNPY/CPON-ir afferents has not been determined in thepresent investigation, but taken together, it seemslikely that the extra-sympathetic NPY/CPONergic fi-bers are contained in the central innervation of thepineal gland or in the parasympathetic innervation.

Processing of proNPY in the Pinealopetal NervesOriginally, NPY was isolated and sequenced from the

porcine brain (Tatemoto et al., 1982), and later thegenes encoding the NPY-precursor (or proNPY) fromrat and human were cloned (Larhammar et al., 1987;Minth et al., 1984). ProNPY, which consists of 69 aminoacid residues, is processed to NPY and a C-terminallypeptide of NPY (CPON) by dibasic cleavage and,subsequently, NPY is amidated at the C-terminal end(Tatemoto et al., 1982). The amide group is essentialfor binding to receptors and thus for NPY’s biologicaleffects (Morley et al., 1987; Sheikh et al., 1989).

It has been shown that nerve fibers in the pinealgland contain NPY(1-36)amide and CPON as two inde-pendent molecules (Mikkelsen et al., 1999). As CPON-immunoreactivity was identified in the pineal gland,but no proNPY (Cozzi et al., 1992; Zhang et al., 1991),the posttranslational processing of proNPY must becompleted before the propeptide reaches the pinealgland. A complete processing was also found in the ratsuprachiasmatic nuclei and the human frontal cortex(Blinkenberg et al., 1990; Mikkelsen et al., 1993). Incontrast, ProNPY has been detected in small amountsin human phaechromocytomas and neuroblastoma celllines (O’Hare and Schwartz, 1989), and in rat anteriorpituitary (unpublished observations). It is postulatedthat enzymes responsible for cleavage of proNPY showdifferent features in different tissues.

The immunohistochemical results indicate that thenumber of NPY (non-amidated) and CPON-ir fiberswas higher than for NPY-amide in the pineal gland asalso seen in the human cerebral cortex (Blinkenberg etal., 1990). The antigenicity of epitopes is influenced byfixation. In the present case, it is reasonable to believethat the small epitope, which only consists of theamidated C-terminal amino acid tyrosine, is moreaccessible for denaturation by the fixative used thanother epitopes examined here. Therefore, the reducedimmunoreactivity observed for Cys-NPY32-36-amide com-pared to the other antigens may be the result of fixationand not any major difference in concentrations betweenthe peptides.

Figs. 22–23. Electron micrograph of NPY-ir nerve terminal be-tween two pinealocytes (Pi) (Fig. 22). Electron micrograph of NPY-irnerve terminals and two sympathetic, non-immunoreactive nerveterminals (arrows) in the perivascular space of the rat pineal gland.Ca, fenestrated capillary (Fig. 23). Scale bars 5 1 µm.


Figs. 22 and 23.

Figs. 24–28. The distribution of NPY-ir nerve fibers in the pineal complex of a bilateral ganglionecto-mized rat. The nerve fibers are present in the deep pineal gland (Fig. 24), the pineal stalk (Fig. 25), and inthe superficial pineal gland (Figs. 26–28). However, only occasionally are fibers found in the superficialpineal gland.


The immunohistochemical results indicate the pres-ence of CPON as a completely processed molecule innerve fibers of the pineal gland, as in the rat suprachi-asmatic nucleus (Mikkelsen and O’Hare, 1991) andCPON has also been found in plasma from patientswith phaechromocytomas (Allen et al., 1987). It is likely

that CPON is secreted together with NPY from theadrenal medulla and probably also from nerve fibers,but its role, if any, as a neurotransmitter or neurohor-mone is at present unknown. In the systems tested todate, CPON has no physiological effects (Potter et al.,1989).

Figs. 29–32. Pairs of photomicrographs showing double fluores-cent immunostained sections from the rat deep (Figs. 30, 32) andsuperficial (Figs. 29, 31) pineal gland. The upper pair shows thedistribution of TH (Figs. 29 and 30), whereas the lower row showsnerve fibers stained for CPON-immunoreactivity (Figs. 31 and 32).

The large arrows point to fibers immunoreactive for only TH in thepineal parenchyma, whereas the small arrows point to a TH-positivefiber in the posterior commissure (pc) running towards the deep pinealgland. Both of these fibers contain TH and not CPON. PR 5 pinealrecess. Scale bar 5 100 µm.


Localization of NPY/CPON-Immunoreactive (ir)Nerves in the Pineal Complex

The localization of the NPY/CPON-ir nerves in thepineal complex were similar to the sympathetic innerva-tion as revealed with histofluorescence and immunohis-tochemical identification of catecholamine-synthesiz-ing enzymes (Nielsen and Møller, 1975; Zhang et al.,1991; present study); i.e., fibers were distributed in allparts of the rat pineal complex. Furthermore, NPY/CPON-ir fibers were found in the meningeal tissue, theadjacent cerebral vessels, and the choroid plexus. At thelight microscopical level, the location of NPY/CPON-irnerves was mostly confined to the connective tissuespaces and the vasculature, but single fibers appearedoften in close opposition to the parenchyma of thesuperficial pineal gland. This ‘‘dual’’ location is similarto the distribution of NPY in other organs, as forexample, in the thyroid and the submandibular gland,where NPY has been found in fibers associated to thevascular system as well as to the endo- and exocrinecells (Leblanc et al., 1987). In the heart, NPYergicnerves are distributed among the myocytes and thecardiac vasculature (Gu et al., 1983, 1984; Mikkelsen etal., 1990; Wharton and Polak, 1990), and are consideredto play a dual role associated with both regulation ofblood flow and contraction of muscle cells (Allen et al.,1983, 1986; Maturi et al., 1989; Svendsen et al., 1990).Similarly, in the pineal gland NPY/CPONergic fiberswere related to both the vessels and the pinealocytesand it seems that NPY also plays a dual role in thepineal gland (see below).

At the ultrastructural level, the immunoreactivitywas observed in nerve fibres and nerve terminals. Mostof the nerve terminals could be identified as sympa-thetic terminals due to the presence of small dense coretransmitter vesicles. Some of the sympathetic termi-nals did not stain for NPY in accord with our resultsobtained by double stainings for TH and NPY/CPON inthis study. The nerve fibres and terminals were locatedin the perivascular spaces but also in the extracellularspaces between the pinealocytes. Synaptic contactsbetween the NPY-ir terminals and the pinealocytes orinsterstitial were never observed. Neither were con-tacts between the immunoreactive terminals and arte-riolar smooth muscle cells or capillary pericytes ob-served. In order to bind to the NPY-receptors onpinealocytes and blood vessels, NPY must diffuse fromthe presynaptic terminals to their target organs, inaccordence with the innervation pattern described inmost endocrine and exocrine gland.

Other neuropeptides playing a role on melatoninmetabolism and secretion show similarities to NPY/CPON. Vasoactive intestinal peptide (VIP), a peptidewith a very potent effect on melatonin secretion (Yuwiler,1983), has also been demonstrated in a perivasularlocation, and to a lesser extent, among the pinealocytes(Mikkelsen, 1989; Cozzi, this issue). The peptidergicinnervation of the pineal gland may, therefore, beregarded as a multineuronal input, which is character-ized by a release of peptides to the extracellular spaces,and therefore influences a group of different cells in thegland.

The deep pineal gland consists of a rather homog-enous population of pinealocytes, and the NPY/CPON-irnerves were intermingled between the pinealocytes.

Fig. 33. The density of immunoreactive fibers for CPON and TH inthe superficial pineal of the rat.

Fig. 34. The relative density of perikarya containing CPON and/orTH immunoreactivity.


This morphology more clearly indicates a direct influ-ence on the endocrine part of the organ. A few NPY/CPON-ir nerves were found in relation to the subarach-noid space and the ventricular ependyma. Aninnervation of the ventricular ependyma has also been

described in the third ventricle (McDonald, et al., 1988;Sabatino et al., 1987), yet the possible release ofNPY/CPON to the cerebrospinal fluid has not beenelucidated. However, administration of NPY in thecerebrospinal fluid elicits a number of neuroendocrineactions (Kaynard et al., 1990; Woller and Terasawa,1991).

Origin of the NPY/CPON-ir Nerves Innervatingthe Pineal Gland

It has previously been shown that approximately97% of the content of NPY disappear in the superficialpineal gland of the rat after superior cervical ganglionec-tomy, showing that the major NPY/CPONergic inputoriginates from the sympathetic nervous system (Mik-kelsen et al., 1999). The origin of the NPYergic innerva-tion has been studied both after ablation of the superiorcervical ganglia (Schon et al., 1985) and with a combina-tion of retrograde tracing and immunohistochemistry(Reuss and Moore, 1989), and both reports concludedthat the NPYergic nerves originated exclusively fromthe sympathetic nervous system. In contrast to thesestudies, we have shown that the sympathetic nervoussystem is not the only source of NPY/CPONergic nervesin the rat pineal gland, especially in the deep pinealgland (Zhang et al., 1991; present study). In addition,we carried out a detailed analysis not only of NPY innormal and ganglionectomized animals, but comparedthe staining for CPON and NPY in the pineal gland,and also in the choroid plexus and the vasculature.Remaining NPY-ir nerves were found in the pinealgland and the pial arterioles, but never observed in thechoroid plexus in the suprapineal recess. At present, webelieve that some of the remaining NPY/CPON ispresent in either the central innervation or a parasym-pathetic innervation or both. This assumption is basedon the observation that most of the NPY/CPON-ir fibersremaining after ganglionectomy were seen in that partof the pineal complex known to receive a centralinnervation, namely the deep pineal gland (Mikkelsenand Møller, 1990).

A parasympathetic innervation of the pineal glandgland is also present (Kenny, 1965; Romijn, 1975; seealso Phansuwan-Pujito et al., this issue). Recent stud-ies have shown the presence of choline acetyl-transfer-ase in intrapineal nerve fibers (Phansuwan-Pujito etal., 1991) and NPY been found to be colocalized withacetylcholine in parasympathetic ganglia known toproject to the pineal gland (Gibbins and Morris, 1988;Leblanc et al., 1987; Shiotani et al., 1986).

The origin of the NPY/CPON-ir neurons contained inthe central pinealopetal projection has not been identi-fied yet with certainty. A NPYergic projection originat-ing from the intergeniculate leaflet of the lateral genicu-late nucleus to the suprachiasmatic nucleus has beendemonstrated (Card and Moore, 1989; Harrington etal., 1985, 1987; Mikkelsen, 1990). This projection hasbeen shown to mediate phase advances to the pace-maker (Albers and Ferris, 1984; Rusak and Bina, 1990;Rusak et al., 1989). Since also the intergeniculateleaflet projects to the pineal gland via the geniculopi-neal projection (Mikkelsen and Møller, 1990), it seemsreasonable to believe that this projection containsNPY/CPON, too, but the definition of the neurochemi-cal content of this projection is still lacking. It is notable

Figs. 35–36. Pair of photomicrographs of a median section throughthe deep pineal gland from a bilateral ganglionectomised rat dual-labeled for TH (Fig. 35) and CPON (Fig. 36). As illustrated, nopositive CPON nerve fibers contained TH and vice versa. Magnifica-tion as in Figure 32.


that the geniculopineal projection is restricted to thedeep pineal gland, and it may be that this part of thepineal complex plays a specific role in pineal regulationand physiology.

NPYergic Innervation DiffersAmong Mammalian Species

It is evident from the available literature that thedistribution of NPY nerve fibers in the mammalianpineal is strongly species dependent. The rat and theovine are species with a large amount of nerve fiberscontaining NPY. In contrast, the primate containsrather little NPY, but a relatively large portion of theNPYergic input appears to originate directly from thebrain (Mikkelsen and Mick, 1992).

Role of NPY in Pineal PhysiologyThe cells possessing NPY (Y) receptors in the pineal

gland are also unknown, but specific binding sites havebeen demonstrated in suspensions of cultured pinealcells (Olcese, 1991), indicating that post-synaptic recep-tors are present in the pineal. In coherence with thefunctional effect of NPY on the synthesis of melatonin,it is speculated that the pinealocytes are the principalNPY-receptors-bearing cells in the pineal gland. Fur-ther evidence has shown that the NPY binding site is ofthe Y1 subtype (Simonneaux et al., 1991), which is alsosupported by revers-transcriptase polymerase chainreaction studies, showing that only Y1 mRNA, and notany of the other subtypes (Y2, Y4, or Y5) were expressed(Mikkelsen et al., 1999).

The role of NPY in peripheral organs has beenattributed to its cotransmission with catecholamines,because they are co-stored in the same postganglioner-gic nerves (Lundberg et al., 1982), and presumably inthe same presynaptic vesicles (Fried et al., 1985). NPYseems to have at least three effects on the sympatheticneuro-effector junction. There is a presynaptic effect(expressed as inhibition of noradrenaline release), andtwo postsynaptic effects: a direct response, and a poten-tiation/inhibition of the noradrenalinergic response (Hå-kanson et al., 1986; Potter et al., 1989; Wahlestedt etal., 1986). In general, NPY and the structurally relatedpeptide YY (PYY) and pancreatic polypeptide (PP) playa role in regulation of the vascular tonus. NPY con-tracts isolated peripheral blood vessels, but the re-sponses in the form of dose-response relationships, aredifferent from organ to organ (Edvinsson et al., 1984,1987). Further, NPY is capable of inhibiting an electri-cally stimulated release of noradrenaline and it ap-pears that NPY may not only be involved in postsynap-tic effect on target cells, but also influences the releaseof noradrenaline from the presynaptic terminal of thesympathetic fiber (Lundberg et al., 1987). Also, NPYseems to have a dual function in many organs. It exertsan effect on the resistance arteries (Joshua, 1991), andon other cells of the organ. With regard to the pinealgland, the role of NPY may be important because thenoradrenergic neurotransmission has for many yearsbeen considered to be essential for regulation of melato-nin secretion (Cardinali et al., 1981; Klein and Moore,1979; Moore and Klein, 1974). Application of NPY innanomolar concentrations inhibits the noradrenergicstimulation of melatonin secretion in vitro (Olcese,1991), but did not seem to have any effect alone

(Morgan et al., 1988a,b; Olcese, 1991; Williams et al.,1989). Other studies in vivo or on pineal slices havebeen more difficult to interpret, since only under cer-tain concentrations (Vacas et al., 1987) and in the darkperiod (Reuss and Schroder, 1987), does NPY seem tohave any effect on either the NAT-activity or themelatonin levels in the medium. If NPY and noradrena-line are secreted concomitantly from the presynapticnerves, NPY has, according to the results of Olcese(1991), the potential to abolish the action of noradrena-line, which is consistent with the results from Kassis etal. (1987), demonstrating that NPY inhibits cAMP viaan inhibitory G-protein. It is shown that NPY inhibitsthe stimulated release of [3H]noradrenaline (Yokoo etal., 1987), but similar studies are still lacking in thepineal gland. Interestingly, another study using theradioactive microsphere technique for measurementsof regional blood flow, has shown that NPY alonereduces the blood flow in the pineal gland of the rabbit,and that this effect is not changed by concommitantapplication with an alpha-blocker (Nilsson, 1991). Theseresults raise interesting questions regarding the role ofthe extrasympathetic NPY-innervation, since this mayhave the potential to play a separate role or to modulatesympathetic transmission as well.

In the European hamster (Cricetus cricetus), a num-ber of NPYergic intrapineal nerve fibers exhibit anannual rhythm with a zenith in midwinter (Møller etal., 1998). At midwinter, 5-methoxytryptophol starts toexhibit a nycthemeral rhythm and the activity ofhydroxyindole-O-methyltransferase (HIOMT), a key en-zyme in the synthesis of melatonin, is significantlyincreased (Ribelayga et al., 1998). In the rat, NPYstimulates HIOMT-activity (Ribelayga et al., 1997). IfNPY also stimulates HIOMT in the European hamster,NPY might be directly involved in the annual regula-tions of the pineal gland in this species.

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Neuropeptide Y in the mammalian pineal gland

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