Ž .Brain Research 755 1997 326–330

Short communication

Ž .The locus coeruleus of the Japanese monkey Macaca fuscata does notexpress m-opioid receptor-like immunoreactivity

Jun Chen a,1, Natsu Koyama a, Takeshi Kaneko b, Noboru Mizuno b,)

a Department of Physiology, Shiga UniÕersity of Medical Science, Seta, Otsu 520-21, Japanb Department of Morphological Brain Science, Faculty of Medicine, Kyoto UniÕersity, Kyoto 606-01, Japan

Accepted 4 February 1997

Abstract

Ž . Ž .It is well known that locus coeruleus LC of the rat shows intense m-opioid receptor-like immunoreactivity MOR-LI . In the courseŽ .of our study on the distribution of MOR-LI in the brain of the Japanese monkey Macaca fuscata , however, no MOR-LI was found in

the LC although the distribution pattern of MOR-LI in other regions of the lower brainstem of the monkey was essentially the same asthat observed in the rat. It was also found that immunoreactivity for Met-enkephalin, the most potent endogenous ligand for MOR, wasintense in the rat LC, but very weak, if any, in the monkey LC. MOR may not be expressed in the monkey LC.

Keywords: m-Opioid receptor; Locus coeruleus; Morphine; Enkephalin; Monkey; Immunohistochemistry

Ž .The locus coeruleus LC , a cluster of noradrenergicneurons, has been reported to be involved in control of

w xvigilance 2,21 , facilitation of ventral horn motoneuronsw x Ž11 and modulation of pain processing for review, seew x.16 . It has also been indicated in the rat that the activityof LC noradrenergic neurons are under control of endoge-nous opioid peptides and highly influenced with morphine,and that the effects of these opioid peptides on LC neurons

Ž . w xare exerted mainly through m-opioid receptor MOR 3,13Ž w x.for review, see 6,22 . In fact, intense expression of MORin the LC of the rat has been indicated by the ligand-bind-

Ž w x.ing autoradiography for review, see 24 , in situ hy-w xbridization histochemistry 20 and immunohistochemistry

w x1,8,19,29,30 . In the LC of the macaque monkey, how-ever, no MOR-binding sites were reported in the previous

w xstudy 18 . There might exist some species differences inthe distribution of MOR in the brain between rat andmacaque monkey. Thus, in the present study, we examined

Ž .the distribution of MOR-like immunoreactivity MOR-LIin the lower brainstem of the Japanese monkey, and com-pared it with that of the rat.

Among the endogenous opioid peptides, methionine5-

) Ž .Corresponding author. Fax: q81 75 753-4340.1 On leave from: the Department of Anatomy, The Fourth Military

Medical University, Xi’an, People’s Republic of China.

Ž .enkephalin Met-ENK is the most potent endogenousligand for cloned MOR though peptides derived from all

w x Žthree opioid peptide family can also bind to MOR 23 forw x.further review, see 7 . Thus, in the present study, the

distribution of Met-Enk in the LC was also examinedimmunohistochemically.

Two Japanese monkeys, one was male weighing 5.7 kgand the other was female weighing 6.7 kg, and three maleSprague–Dawley rats weighing at 250–300 g were used inthe present study. The monkeys, sedated with an intramus-

Ž .cular injection of ketamine hydrochloride 10 mgrkg b.wt ,were anesthetized deeply with an intravenous injection of

Ž .sodium pentobarbital 20 mgrkg b.wt . The rats wereanesthetized deeply with an intraperitoneal injection of

Ž .sodium pentobarbital 100 mgrkg b.wt . The anesthetizedanimals were perfused transcardially with 0.1 M phosphate

Ž .buffer PB, pH 7.3 containing 4% paraformaldehyde,0.2% picric acid and 0.05% glutaraldehyde. Then, thebrains were removed, cut into several blocks and placed in

Ž .0.1 M PB containing 30% wrv sucrose at 48C until theysank. Subsequently, the blocks were cut into frontal sec-tions 40 mm thick on a cryostat. The sections were consec-utively collected and divided into three sets. The first setof the sections were stained with 0.1% Cresyl violet. Thetwo other sets of the sections were immunostained forMOR and ENK, respectively, by avidin-biotin-peroxidase

Ž . w xcomplex ABC method 14 : The sections were incubatedŽ . w xwith a guinea pig anti-MOR antibody 0.5 mgrml 17 or

0006-8993r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved.Ž .PII S0006-8993 97 00191-1

( )J. Chen et al.rBrain Research 755 1997 326–330 327

Žwith rabbit antisera against Met-ENK 1:10 000; Chemi-.con, Temecula, CA, and Inc Star, Stillwater, MN overnight

at room temperature; the antibodies were diluted in 0.01 MŽ .phosphate-buffered 0.9% saline PBS containing 0.5%

Triton X-100, 0.25% l-carrageenan, 0.05% sodium azideand 0.5% normal donkey serum. Subsequently, after arinse with PBS, the sections were further incubated with a

Ž .biotinylated goat anti-rabbit IgG antiserum Vector; 1:250for 2 h, and then incubated with avidin-biotin-peroxidase

Ž .complex Vector; 1:200 for 2 h. Finally, the sections werereacted with 0.05% diaminobenzidine tetrahydrochlorideand 0.001% hydrogen peroxide in 0.05 M Tris-HCl bufferŽ .pH 7.6 . In the control experiments, the primary antibidieswere replaced with normal IgG, or absorbed with the

w xsynthesized peptides as described previously 8,17 ; inthese control experiments no MOR-LI or Met-ENK-LI wasfound.

According to the specifications by Chemicon and Inc-w xStar, Both of the rabbit anti- Met-Enk antisera used in the

present study react partially with leucine5-enkephalinŽ .Leu-Enk . The production and characterization of theanti-MOR antibody which recognized a MOR specific siteŽ .C-terminal residues 369–398 have been described else-

w xwhere 8,17 .To examine if the antibody against rat MOR might

recognize monkey MOR, dot blotting tests were per-formed: In both rat and human, two alternate-splicing

w xvariants of MORs have been cloned; rat MOR1 5,10,25,31w xand rat MOR1B 33 , and human MOR1 and human

w xMOR1A 4,32 . For the dot blotting tests, the peptidescorresponding to partial sequences of rat and human MORs

Ž . Ž .were synthesized Table 1 : rat MOR1 369–398 peptideŽcontains a variant-specific site for rat MOR1 C-terminal

.residues 387–398 and a common site for both rat MOR1Ž .and rat MOR1B C-terminal residues 369–386 ; human

Ž . ŽMOR1 389–400 peptide is identical to rat MOR1 387–. Ž .398 peptide; human MOR1 371–388 is common to

human MOR1 and human MOR1A, and also homologousŽ .to rat MOR1 369–386 with replacement of one amino

Ž 374 372acid Asp in human MOR1 is replaced with Glu in.rat MOR1 . Affinity-purified antibodies to rat MOR1

Ž . w x369–398 were produced in guinea pigs and rabbits 8,17 .Ten nanomoles of synthesized peptides dissolved in dis-tilled water were blotted onto a Zeta Probe membraneŽ .BioRad, Richmond, CA by aspiration with Hybri-Slot

Ž .Manifold Bethesda Research Laboratory, Bethesda, MD .ŽAfter blocking with Block-Ace Dainippon Pharmaceuti-

Fig. 1. Dot blotting tests. Each slot of the membrane was blotted with 10nmol of the synthesized peptide. Each strip was immunostained with the

Ž .guinea pig or rabbit antibody raised against rat MOR1 369–398 .

.cal, Japan , the membrane was incubated for at least 3 hŽ .with the affinity-purified antibody to rat MOR1 369–398

Ž .1 mgrml in 10 mM PBS containing 10% Block-Ace and0.2% Tween 20. The membrane was then incubated for 1 hwith 0.1 mgrml alkaline phosphatase-conjugated anti-

Ž .guinea pig IgG Chemicon, Temecula, CA or anti-rabbitŽ .IgG goat antibody Promega, Madison, WI , and finally

reacted with 0.003% nitroblue tetrazolium and 0.017%bromochloroindolyl phosphate in 100 mM NaCl, 5 mM

Ž .MgCl and 100 mM Tris-HCl pH 9.5 . For absorption2

tests of immunoreactivity, 1 mgrml affinity-purified anti-Ž .body 5.9 pmol IgGrml was incubated for 1 h with a

Ž .20 000-fold excess amount of a peptide 118 nmolrmlbefore reaction with the membrane.

Ž .In the dot immunoblotting tests Fig. 1 , the rabbitŽ .antibody to rat MOR1 369–398 recognized rat MOR1

Ž . Ž387–398 , which was identical to human MOR1 389–. Ž .400 , but not human MOR1 371–388 or rat MOR1

Ž . Ž .369–386 ; the rabbit antibody to rat MOR 369–398recognized the MOR1-specific sequence alone. On theother hand, the guinea pig antibody raised against rat

Ž . Ž . ŽMOR1 369–398 recognized MOR1 387–398 sequence.specific to rat MOR1 and human MOR1 , rat MOR1

Ž . Ž369–386 sequence common to the two splicing variants

Table 1C-terminal amino acid sequences of rat and human m-opioid receptors

Ž . Ž .Rat MOR1 . . . 369 NTREHPSTANTVDRTNHQLENLEAETAPLP 398Ž . Ž .Rat MOR1B . . . 369 NTREHPSTANTVDRTNHQKIDLF 391Ž . Ž .Human MOR1 . . . 371 NTRDHPSTANTVDRTNHQLENLEAETAPLP 400Ž . Ž .Human MOR1A . . . 371 NTRDHPSTANTVDRTNHQVRSL 392

Underlinings indicate the variant specific sites. Outlined capitals point to the difference between rat and human MOR1s.

( )J. Chen et al.rBrain Research 755 1997 326–330328

.of rat MORs, rat MOR1 and rat MOR1B , and also humanŽ . ŽMOR1 371–388 sequence common to human MOR1

.and human MOR1A , although the immunoreactivity forŽ .human MOR1 371–388 was weaker than that for rat

Ž .MOR1 369–386 . The results have indicated that theŽ .guinea pig antibody to rat MOR 369–398 , which was

used for the present immunohistochemical study, recog-Ž .nizes both rat MORs rat MOR1 and rat MOR1B and

Ž .human MORs human MOR1 and human MOR1A . WhenŽ .the antibody to rat MOR1 369–398 was absorbed with

Ž .an excess amount of rat MOR1 369–398 , no immuno-reactivity was detected in the dot blotting tests. Thus, itwas assumed that monkey MOR, including its possiblesplicing variants, was recognized by the guinea pig anti-body used in the present study.

In the pons and medulla oblongata of both the monkeyand rat, intense MOR-LI was seen in the interpeduncularnucleus, parabrachial nuclei, ambiguus nucleus, nucleus of

Žthe solitary tract, and superficial layers lamina I and outer. Žpart of lamina II of the medullary dorsal horn Figs. 2 and

.3 . In these regions, axonal components showing intense ormoderate Met-ENK-LI were also distributed with an in-tense or moderate density.

In the LC, however, an apparent difference was foundin the expression of MOR-LI between the monkey and rat:

Ž .MOR-LI in the LC was intense in the rat Fig. 3b , but

Ž .Fig. 2. MOR-LI in the medullary dorsal horn a and the nucleus of theŽ .solitary tract b of a monkey. MOR-LI is intense in lamina I and outer

Ž .part of lamina II of the medullary dorsal horn a and in the medialŽ .subnucleus of the nucleus of the solitary tract b . MG, magnocellular

part of the medullary dorsal horn; st, solitary tarct; tt, trigeminal tract ofthe spinal nucleus of the trigeminal nerve. Scale bars100 mm.

Fig. 3. Photomicrographs of three adjacent sections through the locusŽ . Žcoeruleus LC and the lateral and medial parabrachial nuclei PBNl and

. Ž . Ž .PBNm of a rat, showing cytoarchitecture a , MOR-LI b and Met-Ž .ENK-LI c . Intense MOR-LI and Met-ENK-LI are seen in both the LC

and the PBN. MES; mesencephalic nucleus of the trigeminal nerve; MT,motor nucleus of the trigeminal nerve; scp, superior cerebellar peduncle.Scale bars300 mm.

Ž .very weak, if any, in the monkey Fig. 4b . Met-ENK-LIŽ .in the LC of the rat was also intense Fig. 3c , whereas thatŽ .in the LC of the monkey was weak Fig. 4c .

The present study confirmed the reported data concern-ing the distribution of MOR-LI in the pons and medulla

w xoblongata of the rat 1,8,19 , and further showed that thedistribution pattern of MOR-LI in the macaque monkeywas essentially the same as that in the rat in the pon-tomedullary regions except for the LC. In the LC, MOR-LIwas intense in the rat, but very weak, if any, in themacaque monkey. The lack of MOR-LI in the monkey LCdid not seem to be due to failures in the immunohisto-

( )J. Chen et al.rBrain Research 755 1997 326–330 329

Ž . Ž .Fig. 4. Photomicrographs of three adjacent sections through the locus coeruleus LC and the lateral and medial parabrachial nuclei PBNl and PBNm of aŽ . Ž . Ž .monkey, showing cytoarchitecture a , MOR-LI b and Met-ENK-LI c . The parabrachial nuclei show clearly both MOR-LI and Met-ENK-LI, whereas

the LC shows neither MOR-LI nor Met-ENK-LI. mes, mesencephalic tract of the trigeminal nerve. Other abbreviations are as in Fig. 2. Scale barss500mm.

chemical techniques, because intense MOR-LI was ob-served in the parabrachial nuclei situated in the LC-con-

Ž .taining sections through the pons of the monkey Fig. 4b .The previous study also reported the lack of MOR binding

w xsites in the LC of the monkey 18 .The rat LC has been reported to contain not only

Met-Enk-immunoreactive fibers, but also Leu-Enk-im-munoreactive and b-endorphin-immunoreactive fibersw x9,26–29 . Among these endogenous opioid peptides, Met-Enk has been known to possess the most strong binding

w x Ž w x.potency for MOR 23 for further review, see 7 . In thepresent study, although the distribution of Met-ENK-im-munoreactive fibers was examined with the rabbit anti-w xMet-Enk antisera which reacted partially with Leu-Enk,the patterns of distribution of the immunoreactive fibersobserved in the pontomedullary regions were in good

Žagreement with those reported previously in the rat forw x. w xreview, see 9,27 and monkey 12,15 : The immuno-

reactivity was intense in the rat LC, while it was veryweak, if any, in the monkey LC.

Thus, the morphological data obtained from the ratsupport the notion that enkephalin and morphine exerttheir effects directly on LC neurons through MOR. On theother hand, the data indicate that both MOR and Met-ENK-containing fibers are scanty, if any, in the LC of the

w xmonkey. Astone-Jones et al. 3 have reported that sys-temic morphine in the awake monkey results in inhibitionof tonic discharge but simultaneously induces a pro-nounced oscillatory discharges of LC neurons, and sug-gested that the decrease in tonic LC discharge might bedue to direct action of morphine on LC neurons, whereaspotentiation of oscillatory discharge of these neurons mightbe due to actions of morphine upon circuits afferent to LC

neurons. However, the present results have suggested thatdirect action of morphine upon neurons in the monkey LC,if exists, might be exerted mainly through opioid receptorsother than MOR.

Acknowledgements

The authors are grateful for the photographic help fromMr. Akira Uesugi. This work was supported in part by theGrants-in-Aid from the Ministry of Education, Science,Culture and Sports of Japan.

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