Light and electron microscopic immunocytochemical study on the innervation of the pineal gland of...

Ž .Brain Research 842 1999 359–375www.elsevier.comrlocaterbres

Research report

Light and electron microscopic immunocytochemical study on theŽ .innervation of the pineal gland of the tree shrew Tupaia glis , with special

reference to peptidergic synaptic junctions with pinealocytes

Masanori Kado a, Akitoshi Yoshida a, Yoshiki Hira b, Yuko Sakai b, Shoji Matsushima b,)

a Department of Ophthalmology, Asahikawa Medical College, Nishikagura, Asahikawa, 078-8510, Japanb Department of Anatomy, Asahikawa Medical College, Nishikagura, Asahikawa, 078-8510, Japan

Accepted 13 July 1999

Abstract

Conventional and immunocytochemical, light- and electron-microscopic studies on the innervation of the pineal gland of the treeŽ . Ž .shrew Tupaia glis were made. Neuropeptide Y NPY -immunoreactive fibers, which were abundantly distributed in the gland,

disappeared almost completely after superior cervical ganglionectomy, suggesting that these fibers are mostly postganglionic sympatheticŽ .fibers. By contrast, tyrosine hydroxylase TH -immunoreactive fibers, which were less numerous than NPY-fibers, remained in

considerable numbers in ganglionectomized animals, indicating the innervation of TH-positive fibers from extrasympathetic sources.Ž . Ž .Bundles of substance P SP - or calcitonin gene-related peptide CGRP -immunoreactive fibers, entering the gland at its distal end, were

left intact after ganglionectomy. SP-fibers were numerous, but CGRP-fibers were scarce in the gland. SP-immunoreactive fibers weremyelinated and nonmyelinated, and were regarded as peripheral fibers because of the presence of a Schwann cell sheath. NPY- andSP-immunoreactive fibers and endings were mainly localized in the pineal parenchyma. NPY-immunoreactive endings synapsedfrequently, and SP-positive ones did less frequently, with the cell bodies of pinealocytes. The results suggest that NPY and SP directlycontrol the activity of pinealocytes. Sections stained for myelin showed that thick and less thick bundles of myelinated fibers entered thegland by way of the habenular and posterior commissures, respectively. Under the electron microscope, the bundles were found to containalso unmyelinated fibers. A considerable number of nerve endings synapsing with the cell bodies of pinealocytes remained inganglionectomized animals; these endings were not immunoreactive for TH or SP. Such synaptic endings may be the terminals ofcommissural fibers. q 1999 Published by Elsevier Science B.V. All rights reserved.

Ž .Keywords: Pineal gland; Synapse; Immunocytochemistry; Peptide; Innervation; Tree shrew Tupaia glis

1. Introduction

A large number of immunohistochemical data gatheredw xsince the pioneering study of Uddman et al. 74 dealing

Ž .with vasoactive intestinal peptide VIP in intrapineal nervefibers in some mammals have demonstrated that nervefibers in the mammalian pineal gland, i.e., peripheral fibersŽ .sympathetic and non-sympathetic and central ones, con-

w xtain a variety of peptides. Since Schon et al. 64 have firstŽ .noticed the presence of neuropeptide Y NPY in sympa-

thetic fibers in the pineal gland of rats, NPY-containingfibers have been found in the pineal gland of many mam-

) Corresponding author. Fax: q81-166-68-2319; E-mail:[email protected]

w xmalian species 46,56 . Non-sympathetic, peripheral fibersŽ .may include substance P SP - and calcitonin gene-related

Ž .peptide CGRP -containing fibers, and VIP-immunoreac-w xtive fibers 46,56 . However, the origins of these fibers are

not fully understood. Extrasympathetic, NPY-immunoreac-tive fibers, probably originating from the central nervoussystem, have been reported to exist in the stalk and the

w xdeep pineal gland of rodents 40,80 and the pineal glandw xof non-rodents 4,42,45 . Retrograde tracings combined

with immunohistochemistry have revealed that SP- andCGRP-immunoreactive fibers originate from the trigeminal

w x w xganglia in gerbils 67 and rats 58 . However, it is alsoassumed that SP-fibers are derived from the central ner-vous system or parasympathetic ganglia in some mammals

w xincluding the rat 44,61,62 . Although VIP-containing fibers

0006-8993r99r$ - see front matter q 1999 Published by Elsevier Science B.V. All rights reserved.Ž .PII: S0006-8993 99 01856-9

( )M. Kado et al.rBrain Research 842 1999 359–375360

are, due to their distribution in the pineal gland and itsw xcapsule, generally assumed to be peripheral fibers 3,35,37 ,

it is determined only in gerbils that these fibers originateŽ . w xfrom the peripheral pterygopalatine ganglia 67 .

Ž .Anti-tyrosine hydroxylase TH is often used to labelpineal sympathetic fibers. After superior cervical gan-glionectomy, however, some TH-immunoreactive fibersremain in the stalk and the deep pineal gland in rodentsw x32,40,80 , and everywhere throughout the gland in non-ro-

w xdents 4 . The remaining TH-fibers are supposed to origi-nate from the central nervous system.

Central fibers from the habenular and posterior commis-sures innervate the pineal gland of many mammalian

w xspecies 15,16,39,76 . Neuronal tracing studies have re-vealed that central fibers are distributed in the proximalregion of the rodent pineal gland, but not in the distalregion, which constitutes the main portion of the organw x39 . On the other hand, it is generally believed that thepineal gland receives a rich supply of central fibers in

w xnon-rodent species 39,76 . Thus, the density of centralfibers in the pineal gland appears to be larger in non-ro-

w xdents than in rodents 39 . In addition to NPY, SP and TH,various peptides such as luteinizing hormone–releasing

Ž . Ž .hormone LH–RH , somatostatin SOM , met- and leu-en-Ž . Ž . Ž .kephalins ENK , oxytocin OXY and vasopressin VAP ,

Ž .and choline acetyltransferase ChAT have been reportedto be present in central fibers in the mammalian pineal

w xgland 15,16,46,53 . However, the previous immunohisto-chemical studies have not succeeded in demonstrating alarge number of central fibers coming from the commis-sures in non-rodent species. In addition, combined tracingand immunohistochemical studies of pinealopetal, centralfibers have not hitherto been conducted on non-rodents.Thus, the origin and nature of central fibers are stillobscure. In view of the difference in the density of centralfibers in the pineal gland, the central innervation may bemore important in non-rodents than in rodents. However,the immunohistochemical data on the innervation of thepineal gland are sparser in non-rodents than in rodents.Moreover, the differentiation between sympathetic andnon-sympathetic fibers by superior cervical ganglionec-tomy has hardly been made in previous studies on non-ro-dents.

Considering the above-mentioned descriptions, attemptsto examine the nature and distribution of peripheral andcentral, pinealopetal fibers in non-rodents may contributemuch to our knowledge of the innervation of the mam-malian pineal gland. Thus, the tree shrew, which is re-

w xgarded as a primitive primate 22 , was used in the presentstudy. A previous electron microscopic study of this ani-mal showed the presence of myelinated fibers in theproximal region of the pineal gland, and two different

w xnerve endings, each synapsing with pinealocytes 10 . Thus,it is expected that immunocytochemical studies at the lightand electron microscopic levels will provide new informa-tion on the nature of a variety of pinealopetal nerve fibers

and synaptic nerve endings in the pineal gland of the treeshrew. The aims of the present investigation are to demon-

Ž . Ž .strate: 1 peripheral sympathetic and non-sympatheticand central fibers in the pineal gland and adjacent struc-tures immunohistochemically using antibodies against TH,ChAT and various neuropeptides, such as NPY, SP, CGRP,VIP, LH–RH, SOM, ENK, OXY and VAP in intactanimals and those subjected to superior cervical gan-

Ž .glionectomy, 2 the distribution of immunolabeled fibersin the pineal gland, its capsule, and the habenular and

Ž .posterior commissures, 3 the course of myelinated fibersto enter the pineal gland and their intrapineal distribution,

Ž .and 4 the nature of myelinated fibers and intrapinealnerve endings by immunoelectron microscopy.

2. Materials and methods

2.1. Animals and surgery

Ž .A total of 16 tree shrews Tupaia glis of both sexes,Žranging in age from 1.5 to 6 years ranging in weight from

.126 to 202 g , were used, in accordance with the Guide-lines for Animal Experiments of Asahikawa Medical Col-lege. The animals were originally raised in CSK ResearchPark in Nagano Prefecture, Japan, and were transferred tothe animal laboratory of Asahikawa Medical College. Theanimals were since then kept under conditions of con-

Ž .trolled lighting LD 12:12; lights on at 0700 h andŽ .temperature 23"28C for at least 1 month before sacri-

fice. Light was provided by cool-white fluorescent lamps,with the intensity at the level of the cages being 150–350lx. The animals were housed individually in steel cagesand were provided standard laboratory cat food with asmall amount of skim milk powder and water ad libitum.

Four animals were subjected to superior cervical gan-glionectomy. The ganglionectomy was performed under

Ž .ketamine hydrochloride 13.5 mgrkg, i.m. and pento-Ž .barbital 47.5 mgrkg, i.p. anesthesia between 0900 and

1200 h 1 week prior to sacrifice. The ganglia, situateddeep to the bifurcation of the common carotid artery, wereexposed and removed bilaterally with fine forceps.

2.2. Histology

Three animals were killed by decapitation at 1300 hunder the same anesthesia as mentioned in Section 2.1.The pineal glands, together with the surrounding braintissue, were rapidly removed, fixed for 20 h in Bouin’sfluid, dehydrated in ethanol, cleared in benzene, and em-bedded in paraffin. The embedded brain tissues were ori-ented so that the pineal gland could be cut sagittally. Serialsections were prepared at a thickness of 8 mm, and werestained alternatively with hematoxylin–eosin and luxol fastblue.

( )M. Kado et al.rBrain Research 842 1999 359–375 361

2.3. ConÕentional electron microscopy

Two unoperated and one ganglionectomized animalswere anesthetized at 1300 h in the same way as describedin Section 2.1., and were perfused through the left cardiac

Žventricle with Tyrode’s solution containing heparin 4000.IUrkg and 2% dextran, followed by a fixative consisting

of 4% paraformaldehyde, 8% glutaraldehyde, and 2% dex-Ž .tran in 0.05 M cacodylate buffer pH 7.4 at room tem-

perature. The perfusion with small amounts of a strongaldehyde fixative containing 8% glutaraldehyde and 4%paraformaldehyde, before perfusing with a main fixativecontaining a lower concentrations of aldehydes, was found

w xto prevent shrinkage of the pineal gland in the rat 65 . Onthe basis of the above observations, we tried to use aconcentrated aldehyde fixative containing 8% glutaralde-hyde and 4% paraformaldehyde only as the perfusate, andfound that the ultrastructure of the pineal gland of the

w xcotton rat was well-preserved 63 . Thus, the same alde-hyde fixative was used in the present study. The pinealglands with the portions of the habenular and posteriorcommissures attached were then dissected out under adissecting microscope and fixed in the same fixative for atotal of 30 min at room temperature. The tissues werepostfixed in 1.33% osmium tetroxide in 0.067 M s-col-

Ž .lidine buffer pH 7.4 for 1 h at 48C, block-stained in 0.5%uranyl acetate for 15 h at 48C, dehydrated and embedded

w xin low viscosity plastic 70 . Thin sections were stainedwith lead citrate for 5 min and examined in a JEM 100Selectron microscope.

2.4. Immunohistology

Five unoperated and two ganglionectomized animalswere anesthetized at 1300 h in the same way as describedin Section 2.1., and were perfused transcardially with 0.1

Ž .M phosphate-buffered saline PBS containing heparin

Ž .4000 IUrkg , followed by ice-cold Bouin’s fixative with-out acetic acid. The pineal glands together with the sur-rounding brain tissue and the dura were removed and fixedin the same fixative for 20 h at 48C, dehydrated in ethanol,and embedded in paraffin in vacuo. Serial sagittal sectionswere prepared at a thickness of 6 mm.

The immunostaining was performed using the avidin–Žbiotin–peroxidase method Vectastain Elite ABC kit, Vec-

.tor, Burlingame, USA . The primary antibodies used in thepresent study were listed in Table 1. A previous studydescribed the presence of pigment-containing cells in the

w xpineal gland of the tree shrew 10 . Our preliminary obser-vations showed that pigment granules were bleached byhydrogen peroxide and sodium phosphate and thus, these

Ž .were melanin unpublished . It is possible that the presenceof melanin interferes with the identification of immunore-active fibers. Thus, sections were at first pre-treated in 3%H O containing 1% Na HPO for 6 h. Thereafter, sec-2 2 2 4

tions were pre-incubated in 1.5% normal goat serum for 30min at room temperature, and then incubated at 48Covernight in the antisera. Sections were incubated with

Žbiotinylated goat anti-rabbit or anti-mouse IgG diluted.1:200 for 1 h at 328C and then with avidin–biotin–per-

Ž .oxidase complex diluted 1:25 for 1 h at 328C. Finally,sections were reacted with 0.01% diaminobenzidine tetra-hydrochloride and 0.01% H O in 0.05 M Tris–HCl2 2

Ž .buffer pH 7.2 for 4 min at 378C.Every fourth section of the pineal gland and adjacent

structures from three unoperated and two ganglionec-tomized animals were stained successively with anti-TH,anti-NPY, anti-SP or anti-CGRP antisera. Camera lucidadrawings of nerve fibers immunoreactive to different antis-era were made at a magnification of =400. Sections fromtwo unoperated animals were used for the immunohisto-chemical detection of other substances.

The staining specificity of the anti-TH, which was usedfor light microscopic immunohistochemistry, anti-NPY and

Table 1Primary antibodies used in the present study. The working dilutions marked with asterisks are for electron microscopy

Antigen Code Source and reference Raised in Working dilution

TH AB152 Chemicon International Temecula, USA rabbit 1:100Uw xTH Dr. I. Nagatsu 48 rabbit 1:20,000

NPY A609rR2R Biogenesis, Poole, UK rabbit 1:6000U1:18,000

w xSP Dr. S. Daikoku 70 rabbit 1:20,000U1:10,000

CGRP CA-08-220 Cambridge Research Biochemicals, Gadbrook Park, UK rabbit 1:10,000LH–RH Institute for Molecular and Cellular Regulation, Gunma University, mouse 1:4000

Maebashi, JapanVIP 20077 Incstar, Stillwater, USA rabbit 1:4000SOM KA18 Milab, Malmo, Sweden rabbit 1:7500¨met-ENK 20065 Incstar rabbit 1:3000leu-ENK AB1974 Chemicon International rabbit 1:4000ChAT AB143 Chemicon International rabbit 1:1000OXY A481rR4V Biogenesis rabbit 1:8000VAP Institute for Molecular and Cellular Regulation, Gunma University rabbit 1:20,000


anti-LH–RH antisera was tested by replacement of theantiserum by normal rabbit serum or PBS. The specificityof the NPY antiserum was also tested by pre-absorption of

Žthe antiserum with synthetic NPY Peptide Institute, 1.mgrml . In these tests, no specific immunostaining was

detected. The specificity of the TH antiserum, which wasused for electron microscopic immunohistochemistry, and

w xSP antiserum has been reported elsewhere 48,72 .

2.5. Immunoelectron microscopy

Two unoperated and one ganglionectomized animalswere under the same anesthesia as described above, per-fused transcardially at 1300 h with 0.1 M PBS containing

Ž .heparin 4000 IUrkg , followed by ice-cold 4% para-Ž .formaldehyde in 0.1 M phosphate buffer pH 7.4 at room

temperature. Since preliminary observations revealed thatthe quality of preservation was good in the materials fixedby perfusion with the above solution, glutaraldehyde wasnot added to the perfusate in the present study. The pinealglands with the portions of the habenular and posteriorcommissures attached were then dissected out and fixed inthe same fixative for 15 h at 48C. Sagittal sections were cut

Žat 40 mm thickness with a Microslicer Dosaka EM,.Kyoto, Japan .

Sections were washed in PBS, pre-incubated in PBScontaining 10% normal goat serum and 1% thyroglobulin

or 1% bovine serum albumin, and incubated in eitheranti-NPY or anti-TH antiserum for 65–70 h or anti-SPantiserum for 20–24 h at room temperature. Specificitycontrols were performed without primary antisera. Afterrinsing in PBS, sections were incubated in goat anti-rabbit

Ž . Ž .IgG diluted 1:200 Vector, Burlingame, USA for 1 h atroom temperature, and incubated in avidin–biotin–per-

Ž .oxidase complex Vector at 1:50 for 1 h at room tempera-ture. Sections were then transferred to 0.1 M PBS andincubated in 0.01% diaminobenzidine tetrahydrochloride

Ž .and 0.01% H O in 0.1 M PBS pH 7.4 for 10 min at2 2

room temperature. Sections were fixed in 1% osmiumŽ .tetroxide in 0.1 M phosphate buffer pH 7.4 for 20 min at

48C. After rinsing in distilled water, sections were stainedin 0.5% uranyl acetate for 20 min at room temperature,

w xdehydrated and embedded in low viscosity plastic 70 .Thin sections were examined in a JEM 100S electronmicroscope.

3. Results

3.1. Gross anatomy of the pineal gland

The pineal gland of the tree shrew is located deep in thebrain, and has been described to be classified as type A

Fig. 1. Parasagittal section of the brain showing topographical relations of the pineal gland and adjacent structures. CA, cerebral aqueduct; C, cerebellum;CC, corpus callosum; HC, habenular commissure; PC, posterior commissure; CS, confluens sinuum; CH, cerebral hemisphere; SSS, superior sagittal sinus;TM, tectum mesencephali; GCV, great cerebral vein; black arrow, suprapineal recess; white arrow, pineal recess; asterisk, third ventricle; black area, pinealgland.


w x76 . However, the observations made by Phansuwan-Pujitow xet al. 52 indicate that the pineal gland of this animal is

composed of the superficial and deep pineal glands, andthe stalk interconnecting them; the superficial pineal glandis elongated, and is situated on the superior colliculi. Thepineal glands used in the present study were variable inlocation and shape, but were regarded, according to the

w xcriterion proposed by Vollrath 76 , as type AB because ofŽtheir larger length than their maximal width Figs. 1, 2, 6

.and 11 . The glands were usually situated between thesuprapineal recess of the third ventricle rostrally and the

Ž .tectum mesencephali caudally Fig. 1 , and appeared, insagittal sections, as an elongated triangle, with the apexpointed dorsocaudally, and the base applied to the thirdventricle, i.e., the pineal recess; the base and the othersides made the two, superior and inferior, angles, whichabutted directly on the habenular and posterior commis-

Ž .sures, respectively Fig. 1 . The base was usually flat andoccasionally depressed slightly. Some pineal glands ap-

Ž .peared spindle-shaped Fig. 2 . The distal region of suchglands extends for a considerable distance between thetectum mesencephali and the great cerebral vein, but itsend did not reach the confluens of the sinuses.

3.2. Myelinated fiber

Examination of sections stained with luxol fast bluerevealed that thick bundles of myelinated fibers from thehabenular commissure ran dorsocaudally into the dorsal

region of the pineal gland toward its distal end; theseŽ .bundles became thinner toward distal levels Fig. 2 .

Moreover, thinner bundles of myelinated fibers entered thepineal gland by way of the posterior commissure, andcoursed along the surface of its ventral region and the

Ž .surface facing the pineal recess Fig. 2 . These fibers couldnot be traced to the distal region of the gland. In addition,some myelinated fibers ran in the meningeal tissue sur-rounding the great cerebral vein; these fibers were traced

Ž .to the distal end of the pineal gland Fig. 2C .Conventional electron microscopic observations re-

vealed the presence of bundles of cross cut or longitudi-nally cut, myelinated and unmyelinated fibers in the pineal

Ž .parenchyma adjacent to the habenular commissure Fig. 3 .Nerve bundles were intermingled with nerve endings,which contained many small non-granulated vesicles and asmall number of large granulated vesicles. Nerve bundles

Ž .lay in contact with glial cells and pinealocytes Fig. 3 .Myelinated fibers were also found in wide perivascular

spaces near the surface of the distal region of the pinealgland. Their myelin sheath were surrounded by theneurilemma. Such myelinated fibers will be described inmore detail in Section 3.5.

3.3. TH-immunoreactiÕe fiber

In unoperated animals, bundles of TH-immunoreactivefibers were seen to run longitudinally along the greatcerebral vein and to enter the pineal gland at its distal end

Fig. 2. Distribution of myelinated fibers in sagittal sections of the pineal gland of an unoperated animal at different levels. All the profiles of myelinatedfibers in each set of three alternate sections at the levels of A, B and C were superimposed. The numbers of serial sections were: 24, 26 and 28 at the levelof A; 50, 52 and 54 at the level of B; and 68, 70 and 72 at the level of C. Myelinated fibers entering the distal end of the gland were indicated by an arrowat the level of C. SO, subcommissural organ. See Fig. 1 legend for additional abbreviations.


Ž .Fig. 4 . The pineal gland of unoperated animals wasrichly innervated by TH-immunoreactive fibers, whichwere usually seen as small bundles or single fibers withvaricosities in the parenchyma. TH-immunoreactive fiberswere more abundant in the ventral region, where thickerbundles of these fibers were often intermingled. TH-posi-tive fibers generally decreased in density toward the dorsal

Žregion, especially near the habenular commissure Fig..5A . In some animals, a number of TH-immunoreactive

fibers lay on the ependymal surface of the pineal recessŽ .Fig. 5A,B, Figs. 6 and 7 . Some TH-fibers were alsofound on the ependyma overlying the habenular commis-sure. The posterior commissure contained a small numberof TH-immunoreactive fibers, but these fibers were seldom

Ž .in the habenular commissure Fig. 5A .Following ganglionectomy, TH-immunoreactive fibers

in the pineal gland decreased remarkably in number, espe-cially in the ventral region; thick bundles of these fibers

Ž .disappeared entirely in this region Fig. 5B . Bundles ofTH-immunoreactive fibers running along the great cerebralvein also disappeared. A considerable number of singlefibers with TH-immunoreactivity remained in the centralregion of the pineal gland of ganglionectomized animalsŽ .Fig. 5B . TH-containing fibers lying on the surface facingthe pineal recess also persisted after the removal of the

Ž .ganglia Fig. 5BFigs. 6 and 7 . Some of these fibersŽ .appeared to enter the pineal parenchyma Fig. 6 . In

ganglionectomized animals, the habenular commissurecontained some TH-immunoreactive fibers, and in theposterior commissure, these fibers were more abundant

Ž .than those in unoperated animals Fig. 5B .

3.4. NPY-immunoreactiÕe fiber

Thick bundles of NPY-immunoreactive fibers, like thoseof TH-fibers, coursed along the great cerebral vein to enterthe distal end of the pineal gland of unoperated animalsŽ .Fig. 8 . In the pineal gland, NPY-positive fibers were

Ž .more abundant than TH-fibers Fig. 5C . NPY-containingfibers were arranged in networks, the meshes of whichcontained individual parenchymal cells or small groups of

Ž .these cells Fig. 8 . As will be mentioned, immunoelectronmicroscopic observations showed that NPY-positive fiberswere scarce in pericapillary spaces. The distribution of

NPY-positive fibers was similar to that of TH-fibers;NPY-fibers appeared to decrease in number toward the

Ž .habenular commissure Fig. 5C . NPY-immunoreactivefibers were absent from the habenular commissure, but

Žonly a few were found in the posterior commissure Fig..5C . No NPY-immunoreactive fibers were observed on the

ependymal surface of the pineal recess.Bundles of NPY-immunoreactive fibers entering the

distal region of the pineal gland were lost following gan-glionectomy. In ganglionectomized animals, NPY-positivefibers almost disappeared from the pineal gland, but a fewremained in its ventral region; the distribution of NPY-fibers in the commissures was similar to that in unoperated

Ž .animals Fig. 5D .

3.5. SP- or CGRP-immunoreactiÕe fiber

Ž .In unoperated and ganglionectomized Figs. 9 and 10animals, small bundles of SP- or CGRP-immunoreactivefibers coursing along the great cerebral vein were seen toenter the pineal gland at its distal end. Examination ofadjacent sections showed that the course of SP-fibers was

Žessentially the same as that of CGRP-fibers Figs. 9 and.10 . SP-immunoreactive fibers, which usually appeared as

single fibers, were scattered in the distal and ventralregions of the pineal gland of unoperated animals; thesefibers were scarce in the vicinity of the habenular commis-

Ž .sure Fig. 11A . There were some SP-positive fibers in theposterior commissure, but almost none were found in thehabenular commissure. CGRP-immunoreactive fibers,which were much fewer than SP-positive fibers, weredistributed in the peripheral region of the pineal gland of

Ž .unoperated animals Fig. 11C . CGRP-immunoreactivefibers were absent in both commissures in unoperated and

Ž .ganglionectomized animals Fig. 11C,D . The density ofSP- or CGRP-positive fibers in the pineal gland appeared

Ž .to increase after ganglionectomy Fig. 11B,D .SP-immunoreactive, myelinated and unmyelinated fibers

were encountered in the capsule over the distal region ofŽ .the pineal gland Fig. 12 and wide pericapillary spaces

Ž .lying near the surface of this region Figs. 13 and 14 .These fibers were ensheathed by the neurilemma, andcontained a considerable number of large granulated vesi-

Ž .cles Fig. 12 . SP-positive fibers and sympathetic fibers

Fig. 3. Myelinated and unmyelinated fibers in a sagittal section of the pineal parenchyma near the habenular commissure of an unoperated animal. P,pinealocyte nucleus. Bars1 mm.

Ž .Fig. 4. Bundles of TH-immunoreactive fibers entering the distal end of the pineal gland PG of an unoperated animal. Bars20 mm.

Fig. 6. Supraependymal and intraparenchymal TH-immunoreactive fibers in a ganglionectomized animal. PR, pineal recess. Bars20 mm.

Fig. 7. TH-immunoreactive fibers on the surface of the ependyma of the PR of a ganglionectomized animal. Unlabeled endings containing granulated andŽ .non-granulated vesicles arrows are also observed. E, ependymal cell nucleus. Bars1 mm.

Fig. 8. Bundles of NPY-immunoreactive fibers entering the distal end of the PG of an unoperated animal. Bars20 mm.


Ž .were surrounded by the same Schwann cells Fig. 13 . Thecells surrounding these myelinated and unmyelinated fiberswere identified as Schwann cells because of their presencein the connective tissue spaces and the paucity of filamentsin their cytoplasm.

3.6. Other nerÕe fibers

Only a few LH–RH-positive fibers were present in theventral region of the pineal gland adjacent to the posteriorcommissure and this commissure itself. Examination of


Ž . Ž . Ž .Fig. 5. TH-immunoreactive A,B and NPY-immunoreactive C,D fibers in parasagittal sections of the pineal glands of unoperated A,C andŽ .ganglionectomized B,D animals. See Figs. 1 and 2 legends for abbreviations.

sections stained with anti-VIP, anti-SOM, anti-met- andleu-ENK, anti-ChAT, anti-OXY or anti-VAP antibodiesfailed to detect any nerve fiber labeled by these antibodiesin the pineal gland.

3.7. NerÕe ending and synaptic formation

Two types of nerve endings were distinguished underŽ .the conventional electron microscope Fig. 15 . The most

common type contained many small non-granulated vesi-cles and a few large granulated vesicles. The small non-granulated vesicles were either round or flattened in shapeŽ .Fig. 15 . Small granulated vesicles, characteristic of sym-pathetic nerve endings, were rare. The minor type of nerveending also contained small non-granulated vesicles andlarge granulated vesicles, but the former vesicles wereexclusively of round form, and the latter ones were more

Fig. 9. SP-immunoreactive fibers in adjacent sections of the pineal gland of a ganglionectomized animal. PG, distal end of the pineal gland. Bars20 mm.

Fig. 10. CGRP-immunoreactive fibers in adjacent sections of the pineal gland of a ganglionectomized animal. PG, distal end of the pineal gland. Bars20mm.

Ž .Fig. 12. SP-immunoreactive fibers with a Schwann cell sheath S, Schwann cell nucleus in the pineal capsule of a ganglionectomized animal. Bars0.5mm.

Ž . Ž . ŽU .Fig. 13. Three SP-immunoreactive nerve fibers arrow heads ensheathed by a Schwann cell S, Schwann cell nucleus in a pericapillary space in theŽ .pineal gland of an unoperated animal. A sympathetic nerve ending arrow , which is non-immunoreactive for SP and contains round and flattened vesicles,

is also covered by the same Schwann cell sheath. Bars0.5 mm.

Fig. 14. SP-immunoreactive myelinated fiber with a Schwann cell sheath in a pericapillary space in the pineal gland of an unoperated animal. C, capillarylumen. Bars0.5 mm.

Ž .Fig. 15. Two different nerve endings embedded in the cell body of a pinealocyte of an unoperated animal. Small clear vesicles in one ending A are eitherŽ .round or flattened in shape, whereas those in the other B are exclusively of round type. Bars0.5 mm.

Ž .Fig. 16. Group of nerve fibers and endings intervening between the cell bodies of pinealocytes P, pinealocyte nucleus of an unoperated animal. TwoŽ .endings containing a mixed population of round and flattened vesicles make synaptic contacts arrows with the cell bodies of pinealocytes. Bars0.5 mm.


Ž . Ž . Ž .Fig. 11. SP-immunoreactive A,B and CGRP-immunoreactive C,D fibers in parasagittal sections of the PGs of the same, unoperated A,C andŽ .ganglionectomized B,D animals as used for the demonstration of TH- and NPY-immunoreactive fibers in Fig. 6. See Figs. 1 and 2 legends for

abbreviations.

Ž .numerous than those in the major type Fig. 15 . Themajor type of nerve ending almost disappeared followingganglionectomy, whereas the number of the minor one wasnot affected by this treatment. Both types of nerve endingswere predominantly encountered in the parenchyma andoften lay close to pinealocytes. Synapses were found be-tween either type of ending and cell bodies of pinealocytesŽ .Figs. 16 and 17 . Synapses were usually of symmetricaltype, but some asymmetrical synapses were also encoun-tered. Synapses were similar in their morphological fea-

tures regardless of the kinds of synaptic nerve endings. Asw xreported previously 10 , the pineal parenchyma of the tree

shrew was partly composed of glial cells and pigment-con-taining cells. No synaptic formations were recognizedbetween nerve endings and parenchymal cells exceptpinealocytes. Nerve cells were not present in the pinealgland of the tree shrew.

Immunoelectron microscopic observations demonstratedthat NPY- or TH-immunoreactive, sympathetic fibers and

Ž .endings often lay among pinealocytes Fig. 18 . These

Fig. 17. Nerve ending packed with numerous small clear vesicles of round shape and considerable numbers of large granulated vesicles forms synapseŽ .arrow with the cell body of a pinealocyte of an unoperated animal. Bars0.2 mm.

Ž .Fig. 18. NPY-immunoreactive nerve fibers or endings arrows in the parenchyma of an unoperated animal. P, pinealocyte nucleus. Bars1 mm.

Ž .Fig. 19. NPY-immunoreactive nerve ending containing round and flattened vesicles forms synapse arrow with the cell body of a pinealocyte of anunoperated animal. Bars0.2 mm.

Fig. 20. Group of nerve fibers and endings, which are mostly immunoreactive for NPY and contain round and flattened vesicles, are localized near aŽ . ŽU .capillary C, capillary lumen in the PG of an unoperated animal. These fibers and endings are partly exposed to a narrow pericapillary space .

Bars0.5 mm.

Ž . Ž .Fig. 21. SP-immunoreactive nerve ending with round vesicles forming synapses arrows with the cell body of a pinealocyte P, pinealocyte nucleus of anunoperated animal. Bars0.2 mm.

Ž .Fig. 22. Nerve endings, which are non-immunoreactive for both TH and NPY and contains round vesicles, make synapses arrows with the cell body of apinealocyte of a ganglionectomized animal. P, pinealocyte nucleus. Bars0.5 mm.


Žendings contained both round and flattened vesicles Fig..19 . Some NPY-positive endings made synaptic contacts

Ž .with cell bodies of pinealocytes Fig. 19 . Synaptic forma-tions between pinealocytes and NPY-immunoreactive end-ings were localized mainly in the ventral region of thepineal gland. Sympathetic nerve fibers or endings im-munoreactive for NPY or TH were occasionally foundnear pericapillary spaces, partly exposed to these spacesŽ .Fig. 20 .

When sections from the pineal glands of ganglionec-tomized animals were stained with anti-SP, anti-TH orboth, only a small population of nerve endings wereimmunoreactive for SP or TH, and many endings remainedunstained with both anti-SP and anti-TH. These stainedand unstained endings were filled with many small roundvesicles and some large granulated vesicles, and werepredominantly localized in the parenchyma. TH-im-munoreactive endings, which were believed to be sympa-thetic because of the presence of flattened vesicles, disap-peared after ganglionectomy. Nerve endings stained with

Ž .anti-SP Fig. 21 and those unstained with both anti-SPŽ .and anti-TH Fig. 22 made synaptic contacts with cell

bodies of pinealocytes. Synapses could not be detectedbetween TH-immunoreactive endings and pinealocytes.Nerve endings immunoreactive for NPY or SP and thoseimmunonegative for both SP and TH did not form synapseswith glial cells or pigment-containing cells.

4. Discussion

4.1. TH- and NPY-immunoreactiÕe fiber

Bundles of TH- and NPY-immunoreactive fibers run-ning along the great cerebral vein in the tree shrew are, inview of the site of their entrance into the pineal gland andtheir disappearance following superior cervical ganglionec-tomy, believed to be the nervi conarii consisted of postgan-

w xglionic sympathetic fibers 1,14,15,30,57,76 . In somerodent species thus far examined, most of TH- and NPY-immunoreactive fibers in the pineal gland have been re-ported to disappear after superior cervical ganglionectomy,

wsuggesting their origin from the ganglia 18,30,31,40,x64,80 . In non-rodent species, however, considerable num-

bers of TH- and NPY-immunoreactive fibers remain fol-lowing ganglionectomy; these fibers are supposed to origi-

w xnate from the central nervous system 4,42,45 . The presentresults indicate that many TH-containing fibers surviveafter ganglionectomy in the pineal gland of the tree shrew;such non-sympathetic TH-fibers are also assumed to arisefrom the brain, as will be discussed in Section 4.3. Takingall these together, it is reasonable to conclude that pinealTH- or NPY-immunoreactive fibers, probably originatingfrom the brain, are more abundant in non-rodents than inrodents. However, the tree shrew differs from the abovenon-rodents in that NPY-positive fibers in the pineal gland

originate almost exclusively from the superior cervicalganglia. The abundance of NPY in sympathetic fibers inthe pineal gland of the tree shrew suggests its functionalimportance.

As shown in the present study, TH- and NPY-im-munoreactive fibers in the pineal gland of the tree shreware more abundant in the distal or ventral regions than inthe proximal or dorsal regions. A similar distribution of

w xsympathetic fibers appears to exist also in some rodent 8w xand non-rodent 13,33,34 species. In this context, it seems

interesting to note that synapses between sympathetic nerveendings and pinealocytes may occur more frequently in thedistal or ventral regions than in the proximal or dorsal

w xregions in the monkey, Macaca fascicularis 19 and treeŽ .shrew this study . In the tree shrew, many myelinated

fibers from the habenular commissure run in the dorsalregion, whereas less numerous myelinated fibers from theposterior commissure are distributed in the ventral region.The number of myelinated fibers decreases at more distallevels. The distribution of sympathetic fibers may be in-versely related to that of intrapineal commissural fibers.

4.2. SP- and CGRP-immunoreactiÕe fiber

SP- or CGRP-immunoreactive fibers have been demon-wstrated in the pineal gland of several rodent 28,30,58,

x w x62,66,67 and non-rodent 44,61 species. Immunohisto-chemical labeling of these peptidergic fibers combinedwith determination of their origin using retrograde tracingtechniques reveals that SP- and CGRP-containing fibers in

w x w xthe pineal gland of gerbils 67 and rats 58 have thecommon source, i.e., the trigeminal ganglia. In addition,

w xour previous 30 and present studies of serial sectionssuggest the presence of both peptides in the same fibers inthe pineal gland of cotton rats and tree shrews. Thus, in thepresent study, SP and CGRP-positive fibers are classifiedas the same group of fibers. CGRP-fibers are generally

w xmore abundant than SP-fibers in rodents 28,30,66,67 ,whereas in the tree shrew, the converse relationship isobserved between both fibers. Species differences mayexist in the content of both peptides in the same fibers.

SP- or CGRP-immunoreactive fibers in the pineal glandof some mammals are apparently smaller in number than

w xsympathetic fibers 30,67 . This is also true for the treeshrew. Although SP-containing fibers are evenly dis-

w x w xtributed in the pineal gland of cows 44 and monkeys 61 ,these fibers are less numerous in the dorsal region of thepineal gland adjacent to the habenular commissure in thetree shrew. Thus, SP-immunoreactive fibers are similar indistribution to sympathetic fibers in the pineal gland of thisanimal. The distribution of SP-immunoreactive fibers mayalso be related to the uneven distribution of intrapinealcommissural fibers.

We previously found that the pineal gland of the cottonrat was characterized by the supply of very rich CGRP-

w xpositive fibers and less abundant SP-containing fibers 30 .


Careful examination of serial sections strongly suggeststhat the nervi conarii consisting of SP- or CGRP-im-munoreactive fibers and TH- or NPY-positive, sympatheticfibers enter the gland at its distal end, and SP and CGRP,like TH and NPY, coexist in the same fibers. It is assumedthat SP- or CGRP-fibers are not derived from the centralnervous system because of their absence from the habenu-lar and posterior commissures. In addition, the nervi conariiand the pineal gland of the cotton rat contain many myeli-nated fibers, which exhibit a distribution similar to that of

w xSP- or CGRP-fibers 29 . Similar observations are alsow xobtained in the Chinese hamster 28 . Thus, SP- or CGRP-

fibers and myelinated fibers in the nervi conarii and thepineal gland of both species may belong to the same group

w xof fibers 28,30 .The present data indicate that the distribution of SP- or

CGRP-immunoreactive fibers in the pineal gland and themeningeal tissue in the tree shrew is similar to that in thecotton rat and Chinese hamster. In the tree shrew, SP- orCGRP-fibers entering the pineal gland at its distal endmay, in view of their course, constitute part of the nerviconarii. The present study demonstrates that SP-containingfibers in the pineal gland of the tree shrew are partlymyelinated, and have the characteristics of peripheral fibersbecause of having a Schwann cell sheath. It is postulatedthat SP-immunoreactive fibers in the pineal gland of ratsw x w x w x62 , monkeys 61 and cows 44 come from the habenularor posterior commissure. However, since the commissuresin the tree shrew contain very few SP- or CGRP-fibers ornone at all, these fibers may not have their origin in thecentral nervous system. As mentioned, SP- or CGRP-fibersin the pineal gland of some rodents are found to originate

w xfrom the trigeminal ganglia 58,67 . Taking into accountthe morphological features of SP- or CGRP-fibers men-tioned above, this ganglia may be the most probable sourceof these fibers in the pineal gland of the tree shrew.

As shown in the present study, SP- and CGRP-im-munoreactive fibers in the pineal gland of the tree shrewappeared to increase in number following superior cervicalganglionectomy. We observed a similar increase in thenumber of SP- and CGRP-immunoreactive fibers in gan-glionectomized Chinese hamsters, and speculated that thisincrease might be caused by nerve growth factor-related

w xmechanisms 28 . When examining the intrapineal distribu-tion of certain extrasympathetic fibers, the possibility thatthe number of these fibers in question may change inresponse to sympathetic denervation should be considered.

4.3. Commissural fiber

It has long been known, from the observations ofsilver-impregnated sections, that deeply situated pinealglands of various mammals including man are invaded bynerve fibers from the habenular and posterior commissuresw x15,16 . In addition, conventional electron microscopicstudies have demonstrated the localization of central,

myelinated and unmyelinated fibers in the deep pinealgland adjacent to the commissures in several rodent speciesw x15,16 . As shown in the present study, sections stained formyelin reveal that bundles of myelinated fibers enter thepineal gland from the commissures in the tree shrew.

Immunohistochemical studies have shown that nervefibers probably originating from the central nervous sys-tem in the pineal gland of some mammals contain various

w x w xpeptides, i.e., OXY or VAP 2,32,49,51 , SOM 41 , ENKw x w x43 and LH–RH 32,54 . In the present study, intrapinealnerve fibers were stained only with the anti-LH–RH amongthe antibodies against the above peptides. Only a fewLH–RH-containing fibers were present in the pineal glandand the posterior commissure, but almost none were foundin the habenular commissure. In ganglionectomized treeshrews, there were some SP- or TH-immunoreactive fibersin the posterior commissure, but these fibers were rare inthe habenular commissure. Consequently, we were notable to label abundant myelinated and unmyelinated fibersentering the pineal gland from both the habenular andposterior commissures in the tree shrew. The nature ofintrapineal commissural fibers will be considered in afuture study.

It has recently been shown that the pineal gland of thetree shrew contains a moderate number of leu-ENK-im-munoreactive fibers; these fibers are, according to theirdistribution, considered to be pinealopetal fibers mostlyoriginating from the peripheral ganglia and partly from the

w xbrain 52 . The discrepancy between the above data andthose of the present study may be due to the differences inthe affinity of the antibody or the techniques.

We failed to demonstrate ChAT-immunoreactive fibersin the pineal gland of the tree shrew. The existence ofintrapineal nerve fibers immunoreactive for ChAT hasbeen documented for the cow; these fibers are supposed tooriginate from the parasympathetic ganglia or the brainw x53 . A cholinergic innervation may be of little importancein the pineal gland of the tree shrew. This assumption maybe compatible with the observation that intrapineal nervefibers of this animals are devoid of VIP.

The observation on the tree shrew that TH-immunoreac-tive fibers appear to increase in number in the com-missures suggests that the number of extrasympatheticTH-fibers, like that of SP- or CGRP-fibers, increases inresponse to sympathetic denervation. Thus, it cannot bedenied that intrapineal TH-positive fibers that survive afterganglionectomy are commissural fibers, though TH-fibersare scarce in the commissures in unoperated tree shrews.There is another possibility as regards the origin of non-sympathetic TH-immunoreactive fibers. The present studyshowed that TH-positive fibers overlying the ependymalsurface of the pineal recess were left intact and seemed tobe continuous with intraparenchymal TH-fibers in gan-glionectomized tree shrews. This result suggests thatsupraependymal TH-fibers innervate the pineal gland ofthis animal. Scanning electron microscopic observations


revealed the presence of extensive networks of nerve fibersoverlying the ependyma of the pineal recess of some

w x w xrodents 59,77,78 and the brush-tailed possum 73 . Thesupraependymal nerve fibers in the pineal recess were

w xfound to be serotoninergic in some rodents 21,71 andw xNPYergic in the monkey, M. fascicularis 36 . Further

studies are necessary to support the view that supraependy-mal TH-fibers innervate the pineal gland of the tree shrew.

4.4. NerÕe ending and synaptic formation

Sympathetic nerve endings in the pineal gland of thetree shrew were found to contain only a few small gra-nulated vesicles, which are generally used as the mostreliable criterion for identifying these endings under the

w xelectron microscope 24 . Fortunately, however, pinealsympathetic nerve endings of this animal often containsmall clear vesicles of flattened shape. By contrast, smallclear vesicles found in non-sympathetic nerve endingswere exclusively of the round type. The presence of theflat vesicles in pineal sympathetic nerve endings has also

w xbeen reported in some mammals 19,20,24 . The conven-tional electron microscopic data obtained from the treeshrew were confirmed by the immunoelectron microscopicobservations that flat vesicles are present only in NPY- orTH-immunoreactive, sympathetic nerve endings; TH- orSP-immunoreactive nerve endings and those non-im-munoreactive for TH or SP, both of which persist afterganglionectomy, contain only round vesicles. Although thereason why flat vesicles occur only in sympathetic nerveendings remains unknown, these vesicles may be an impor-tant indicator of sympathetic nerve endings.

The presence of synapses between sympathetic nerveendings and pinealocytes has previously been reported in

w xsome mammals including the tree shrew 9,10,19,20,23 .In these conventional electron microscopic studies, theidentification of sympathetic nerve endings was made onlyon the basis of their ultrastructure, and, with the exception

w xof the only study 19 , superior cervical ganglionectomywas not performed to determine if nerve endings in ques-tion are derived from this ganglia. In order to distinguishbetween sympathetic and non-sympathetic nerve endings,we considered, in the present study, their ultrastructure andimmunocytochemical nature, and the effects of superiorcervical ganglionectomy on these endings. As a result, itwas found that sympathetic and non-sympathetic nerveendings were mostly encountered within the parenchyma,and synapsed with the cell bodies of pinealocytes, but notwith the other parenchymal cell types. The present study isthe first to demonstrate synapses between NPY- or SP-im-munoreactive nerve endings and pinealocytes in the mam-malian pineal gland. A recent immunoelectron microscopicstudy has reported the existence of synaptic-like contactsbetween nerve endings containing C-terminal flanking pep-

w xtide of NPY and pinealocytes in the pig 55 . However,since superior cervical ganglionectomy was not done in the

above study, the nature of the synaptic nerve endingsmentioned is not exactly known. The present results indi-cate that in the pineal gland of the tree shrew, NPY-im-munoreactive, synaptic nerve endings originate almost ex-clusively from the superior cervical ganglia, synapsesbetween these endings and pinealocytes are typical, andpostsynaptic structures are apparently pinealocytes.

It has previously been shown, by conventional electronmicroscopic studies, that nerve endings containing smallclear vesicles make synaptic contacts with pinealocytes in

w xseveral rodent 17,27,38 and non-rodent species includingw xthe tree shrew 10–12 . However, the exact nature and

origin of these synaptic nerve endings have not beendetermined. Such synaptic endings in the pineal gland ofthe tree shrew may include SP-nerve endings or those ofcommissural fibers, which will be mentioned below. Asstated earlier, it is tentatively assumed that SP-im-munoreactive, synaptic endings are derived from thetrigeminal ganglia. Anterograde tracing combined withimmunohistochemistry may be useful to confirm this as-sumption.

In a previous study, two types of synaptic nerve end-ings, aminergic and cholinergic, were distinguished in thepineal gland of the tree shrew according to their finestructure; nerve endings, which were assumed to be cholin-

w xergic, contained small clear vesicles 10 . As shown in thepresent observations, considerable numbers of synapticnerve endings remained after superior cervical ganglionec-tomy; these endings contain small clear vesicles of roundshape. The immunoelectron microscopic data from gan-glionectomized animals indicate that some non-sym-pathetic nerve endings are immunoreactive for SP or TH,but the majority of them are non-immunoreactive for both.Since large numbers of myelinated and unmyelinated fibersenter the pineal gland from the commissures in the treeshrew, it is highly probable that non-sympathetic nerveendings containing neither SP nor TH are terminals ofcommissural fibers. The observation that such endingsoften synapse with pinealocytes suggests their functionalimportance. Further immunohistochemical investigationsare needed to determine transmitter substances in intrap-ineal synaptic terminals of commissural fibers in the treeshrew.

4.5. Functional consideration

The present conventional and immunocytochemicalelectron microscopic observations revealed that in thepineal gland of the tree shrew, two kinds of peripheralfibers, i.e., NPY- or TH-immunoreactive, sympatheticfibers and SP- or CGRP-immunoreactive, non-sympatheticfibers, were mostly distributed among pinealocytes; only afew were localized in pericapillary spaces. This distribu-tion pattern of peripheral fibers is in marked contrast tothat in the superficial pineal gland of rodents, where thesefibers are preferentially localized in pericapillary spaces or


w xadjacent to capillaries 25,26,30 . In the tree shrew, unlikein rodents, neuropeptides from these fibers may not playan important role in the regulation of pineal blood flow.The presence of synapses between NPY- or SP-im-munoreactive fibers and pinealocytes in the tree shrewstrongly suggests that these peptides directly control theactivity of pinealocytes of this animal.

Effects of NPY on functional activity of the pinealgland have so far been examined mainly in rats. Theresults from the in vitro experiments showed that in cul-tured pineal glands or pinealocytes, NPY inhibited the

w xrelease of noradrenaline 69,75 or noradrenaline-inducedw xcAMP accumulation 7,50,69 , but there was no agreement

concerning the effect of NPY on melatonin synthesis andrelease. Noradrenaline-induced melatonin release was ei-

w x w xther stimulated 69,75 or inhibited 50 by NPY. In addi-tion, NPY has recently been demonstrated to stimulatehydroxyindole-O-methyltransferase activity and melatonin

w xrelease in cultured pinealocytes of the rat 60 . In thesheep, the only species thus far examined except the rat,noradrenaline-induced melatonin release was reported to

w xbe uninfluenced by NPY 47,79 . Biochemical studies onthe role of SP in the mammalian pineal gland are scarce.SP-binding sites have been demonstrated in the bovine

w xpineal gland 6 . Adenylate cyclase activity in the variousregions including the pineal gland of humans was stimu-

w xlated by SP 5 , whereas in the rat SP failed to affectcAMP accumulation and basal or noradrenaline-stimulated

w xN-acetyltransferase activity in the pineal gland 68 . It isbecoming more and more obvious that NPYergic andSPrCGRPergic, peripheral fibers innervate the pineal glandof mammals in general. In order to make clear the signifi-cance of such peripheral innervation, biochemical studieson the role of these peptides in the pineal gland need to bedone in more mammalian species.

Acknowledgements

We would like to thank Dr. Shigeo Daikoku and Dr.Ikuko Nagatsu for supplying the SP and TH antisera,respectively.

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