d-Opioid Receptor Is Present inPresynaptic Axon Terminals in the Rat

Nucleus Locus Coeruleus: RelationshipsWith Methionine5-Enkephalin

E.J. VAN BOCKSTAELE,1* K. COMMONS,2 AND V.M. PICKEL3

1Department of Pathology, Anatomy, and Cell Biology, Jefferson Medical College of ThomasJefferson University, Philadelphia, Pennsylvania 19107

2Laboratory of Neurobiology and Behavior, The Rockefeller University,New York, New York 10021

3Department of Neurology and Neuroscience, Cornell University Medical College,New York, New York 10021

ABSTRACTThe three classes of opioid receptors, µ, d, and k, are distributed within the locus coeruleus

(LC) of the rat brain. We have recently shown with immunoelectron microscopy that theµ-opioid receptor (µOR) is localized prominently to extrasynaptic sites on the plasmamembranes of noradrenergic perikarya and dendrites of the LC. To further characterize thecellular distribution of other opioid receptors in this region, in this study, we examined theultrastructural localization of an antipeptide sequence unique to the d-opioid receptor (dOR)in sections that were also dual labeled for methionine-enkephalin (M-ENK), an opioid peptideknown to be an endogenous ligand of the dOR. Immunoperoxidase labeling for dOR waslocalized primarily to the plasma membranes of presynaptic axon terminals and was alsoassociated with large dense core vesicles. The dOR-labeled axon terminals formed bothexcitatory (asymmetric) and inhibitory (symmetric) type synaptic specializations with unla-beled dendrites and were frequently apposed by astrocytic processes. Dual labeling showedthat, of 180 dOR-labeled axon terminals, 16% showed colocalization with M-ENK. Theseformed both types of synaptic junctions. Peroxidase labeling for dOR was also observedoccasionally within dendrites, unmyelinated axons, and glial processes. The dOR-labeleddendrites were usually postsynaptic to unlabeled axon terminals that contained both smallclear and large dense core vesicles. These results provide the first ultrastrucutral evidencethat, in the LC, dOR may play a role in the presynaptic modulation of release of bothexcitatory and inhibitory neurotransmitters. They also suggest involvement of dOR inautoregulation of M-ENK release from axon terminals in this region. J. Comp. Neurol.388:575–586, 1997. r 1997 Wiley-Liss, Inc.

Indexing terms: norepinephrine; drug abuse; enkephalin; opiate, morphine

The endogenous opioid peptides, methionine5- and leu-cine5-enkephalin, dynorphin, and b-endorphin, and theopioid receptors (ORs), µ, d, and k, are distributed withinthe locus coeruleus (LC) in the rostral dorsal pons (Eldeand Hokfelt, 1993; Khachaturian et al., 1983; Van Bocks-taele et al., 1995, 1996b). Autoradiographic (Waksman etal., 1986; Sharif and Hughes, 1989; Delay-Goyet et al.,1990; Mansour and Watson, 1993; Mansour et al., 1995) aswell as immunocytochemical studies (Arvidsson et al.,1995) have shown that the dOR is moderately localized inthe LC. However, such techniques did not afford theresolution necessary for determining potential subcellular

differences in the localization of this opioid receptor sub-type or its relation to endogenous opioid peptides. Thisinformation is critical for the LC, because mismatch

Grant sponsor: National Association on Drug Abuse; Grant numbers: R29DA09082, RO3 DA10450; Grant sponsor: NIH, National Institute on MentalHealth; Grant numbers: MH40008, MH00078; Grant sponsor: American HeartAssociation Established InvestigatorAward.

*Correspondence to: E..J. van Bockstaele, Department of Pathology,Anatomy, and Cell Biology, Thomas Jefferson University, 1020 LocustStreet, 520 Jefferson Alumni Hall, Philadelphia, PA 19107. E-mail:vanbock1@jeflin.tju.edu

Received 11 March 1997; Revised 29 May 1997; Accepted 12 June 1997

THE JOURNAL OF COMPARATIVE NEUROLOGY 388:575–586 (1997)

r 1997 WILEY-LISS, INC.

between opioid ligands and their receptors has beendescribed in other brain regions (Herkenham, 1987; Chenget al., 1995).

Physiological and pharmacological studies have indi-cated that opioid peptides as well as exogenous opiates,such as morphine, affect the discharge rate of LC neurons(Bird and Kuhar, 1977; Pepper and Henderson, 1980;Harris and Williams, 1991; Alreja and Aghajanian, 1993).Electrophysiological studies have indicated that manyeffects of endogenous or exogenous opiates act primarily atthe µ subtype (Williams et al., 1982; North and Williams,1985) in the LC. Such analyses suggested that the µOR islocated postsynaptically, based on studies showing thatthe hyperpolarization of intracellularly recorded LC neu-rons caused by opioid peptides is associated with a fall ininput resistance that persists under conditions in whichsynaptic transmission is blocked (North and Williams,1985; North et al., 1987). By using immunoelectron micros-copy, we have recently shown that the µOR is localizedprominently to extrasynaptic portions of the plasma mem-branes of noradrenergic perikarya and dendrites (VanBockstaele et al., 1996a), confirming the predictions of theelectrophysiological experiments. We also showed thatµOR immunoreactivity is found in dendrites that areapposed by axon terminals containing the opioid peptideleucine5-enkephalin (Van Bockstaele et al., 1996b). Incontrast to µOR activation, actions of ligands at the dreceptor, such as D-Pen-D-Pen-enkephalin (DPDPE; Northet al., 1987), have been shown to affect the physiologicalactivity of LC neurons by presynaptic modulation oftransmitter release (North et al., 1987; Ronken et al.,1993). This is similar to mechanisms reported for the kreceptor (McFadzean et al., 1987). Presynaptic modulationmediated by dORs has been shown in several other brainregions (Zajac et al., 1989; Collin et al., 1991), includingthe spinal cord and the diencephalon.

Although electrophysiological experiments suggest thatdORs are presynaptic in the LC, there is no ultrastructuralevidence to date that supports this contention. Therefore,in this study, we examined the ultrastructural localizationof an antipeptide antibody generated against a specificpeptide sequence uniquely present in the dOR (Cheng etal., 1995) with the detection of an antibody to the opioidpeptide methionine-enkephalin (M-ENK) in the same sec-tion of tissue. The region selected for ultrastructuralanalysis is known to contain the noradrenergic cell bodiesof the LC. These results provide the first ultrastructuraldemonstration that the dOR is localized extensively toaxon terminals, some of which contain immunolabeling forM-ENK in the LC. Immunolabeling for dOR in these axonterminals was commonly associated with the plasma mem-branes of terminals as well as with dense core vesicleswithin the axoplasm. Finally, dOR immunoreactivity wasdetected occasionally in dendrites, unmyelinated axons,and astrocytic processes.

MATERIALS AND METHODS

Tissue preparation

The experimental protocol used in the present study wasapproved by the Institutional Animal Care and Use Com-mittee, Department ofAnimal Resources, at Thomas Jeffer-son University. Two adult male Sprague Dawley rats(Taconic Farms, Germantown, NJ; 200–250 g) were used.Animals were deeply anesthetized with sodium pentobar-

bital and were perfused transcardially through the ascend-ing aorta with 50 ml of 3.8% acrolein (Polysciences,Warrington, PA) and 200 ml of 2% paraformaldehyde in 0.1M phosphate buffer (PB), pH 7.4. Immediately followingperfusion-fixation, the brains were removed, cut into 1–3mm coronal slices, and placed in the same fixative for anadditional 30 minutes. Forty-micron–thick sections werecut through the rostrocaudal extent of the LC with aVibratome and collected into 0.1 M PB.

Antisera specificity

The characterization and specificity of the guinea pigantiserum against the dOR (Immuno-Dynamics, La Jolla,CA) and the rabbit antibody against M-ENK (Incstar,Stillwater, MN) have been described previously (Cheng etal., 1995; Van Bockstaele et al., 1995). The guinea pigpolyclonal antiserum was raised against peptides 34–47(p34), an amino acid sequence within the extracellularN-terminus of the dOR recently cloned from mouse neuro-blastoma glioma (NG-108) cells (Cheng et al., 1995).Immunolabeling was selectively adsorbed with the appro-priate peptide with concentrations of 1 and 10 µg/ml.Immunodot-blot analysis (Cheng et al., 1995) was alsoused to show specificity of the guinea pig antiserumagainst the dOR peptide. The M-ENK antibody was shownto recognize primarily M-ENK, and not leucine5-enkepha-lin or dynorphin A (Cheng et al., 1995). The immunoreac-tion product was shown to be abolished in tissue sectionsthrough the brainstem by preadsorption of the M-ENKantibody with high concentrations of M-ENK (Svingos etal., 1995; Van Bockstaele et al., 1996b). To evaluate thepossible recognition of the primary guinea pig antiserumby secondary antibodies against rabbit immunoglobulins(IgGs) in the dual-labeling experiments, some sectionswere processed for dual labeling with omission of therabbit antiserum.

Immunocytochemical labeling

The Vibratome sections were placed for 30 minutes in1% sodium borohydride in 0.1 M PB to remove reactivealdehydes (Leranth and Pickel, 1989). Sections were thenrinsed extensively in 0.1 M Tris-buffered saline (TBS) andincubated for 30 minutes in 0.5% bovine serum albumin(BSA) in 0.1 M TBS for 30 minutes prior to the primaryantibody incubation. Tissue sections containing the LCwere incubated in a mixture of guinea pig anti-dORantibody (1:2,000; Cheng et al., 1995) and a rabbit monoclo-nal anti-M-ENK antibody (1:20,000; Incstar, Stillwater,MN). Alternate sections were incubated with preadsorbedcontrol antiserum. The control serum was prepared by theaddition of p34 peptide (500 µg/ml) to the primary dORantiserum. Sections were treated with 0.3% Triton X-100for light microscopy but lacked Triton X-100 for electronmicroscopy.

Methods for dual immunocytochemical labeling havebeen described previously (Chan et al., 1990; Van Bocks-taele et al., 1994). For all incubations and washes, sectionswere continuously agitated with a Thomas rotator (FisherScientific, Pittsburgh, PA). The dOR was immunolabeledby using the immunoperoxidase method, and M-ENK wasidentified with the immunogold-silver labeling method forultrastructural observations. Tissue sections were incu-bated in the primary antibody 15–18 hours at roomtemperature. They were then rinsed three times in 0.1 MTBS and incubated at room temperature for 30 minutes in

576 E.J. VAN BOCKSTAELE ET AL.

biotinylated goat anti-guinea pig (1:400; Amersham Corp.,Arlington Heights, IL) followed by incubation in avidin-biotin peroxidase complex (ABC, Vector Laboratories, Bur-lingame, CA; 1:200; Hsu et al., 1981) for immunoperoxi-dase labeling of the dOR. The dOR was then visualized by a4-minute reaction in 22 mg of 3-38-diaminobenzidine (Al-drich, Milwaukee, WI) and 10 µl of 30% hydrogen peroxidein 100 ml of 0.1 M TBS.

For gold-silver labeling of M-ENK, sections were rinsedin 0.01 M phosphate-buffered saline (PBS) and incubatedin a solution of 0.01 M PBS containing 0.1% gelatin and0.8% BSA for 30 minutes. Sections were then incubated ina goat anti-rabbit IgG conjugated to 1 nm gold particles(Amersham Corp.) for 2 hours at room temperature. Thesewere rinsed in 0.01 M PBS containing the same concentra-tions of gelatin and BSA described above and were subse-quently rinsed with 0.01 M PBS. Sections were thenincubated in 1.25% glutaraldehyde in 0.01 M PBS for 10minutes, followed by a wash in 0.01 M PBS, and then in 0.2M sodium citrate buffer, pH 7.4. Silver intensification ofthe gold particles was achieved by using a silver-enhance-ment kit (Amersham Corp.). The optimal silver enhance-ment times were determined empirically for each experi-ment and ranged from 7 to 8 minutes for electronmicroscopy and from 11 to 12 minutes for light microscopy(Chan et al., 1990).

For light microscopy, sections were rinsed in 0.01 M PB,mounted onto gelatin-coated glass slides, air dried, andcoverslipped in DPX (Aldrich). For electron microscopy,sections were rinsed in 0.1 M PBS, incubated in 2%osmium tetroxide in 0.1 M PB for 1 hour, washed in 0.1 MPB, dehydrated, and flat embedded in Epon 812 (Leranthand Pickel, 1989). Thin sections of approximately 55–65nm were cut from the outer surface of the tissue with adiamond knife (Diatome, Fort Washington, PA) by usingan RMC ultramicrotome. These were collected on gridsand counterstained with uranyl acetate and Reynolds leadcitrate.

Data analysis

Thin sections of tissue prepared for electron microscopywere selected immediately adjacent to the fourth ventriclecorresponding to plate 50 of the rat brain atlas of Swanson(1992). Analysis was performed on thin sections collectedsufficiently close to the outer surface of the tissue to permitdetection of both dOR and M-ENK immunoreactivities.The classification of identified cellular elements was basedon Peters et al. (1991). Structures were defined as beingproximal dendrites if they contained endoplasmic reticu-lum and were larger than 0.7 µm in diameter. Axonterminals were distinguished from unmyelinated axonsbased on their content of synaptic vesicles and diametersgreater than 0.1 µm. A terminal was considered to besynaptic when it showed a junctional complex, a restrictedzone of parallel membrane apposition with slight enlarge-ment of the intercellular space, and/or associated postsyn-aptic thickening. Asymmetric synapses were identified bythe presence of thick postsynaptic densities (Gray’s type I;Gray, 1959); symmetric synapses, on the other hand, hadthin densities (Gray’s type II) both pre- and postsynapti-cally. Nonsynaptic contacts, or appositions, were definedby closely spaced, parallel plasma membranes of immuno-reactive axons and other axon terminals or dendrites.These lacked recognizable specializations and were notseparated by glial processes.

Tissue sections with the best immunocytochemical label-ing and preservation of ultrastructural morphology wereincluded in the analysis. At least ten grids containing fiveto ten thin sections each were collected from the surface ofthree or more plastic-embedded sections containing the LCfrom each animal. Photographs were taken only when bothmarkers were clearly in similar portions of the neuropil infields magnified 37,000.

Criteria for determining gold-silverlabeling of profiles

To ensure specificity in the preembedding gold-silverlabeling method, selective gold-silver-labeled profiles wereidentified by the presence in single thin sections of at leastthree to four gold particles within membrane-enclosedstructures. This differed from ‘‘spurious’’ gold-silver label-ing, which appeared as isolated deposits of gold-silverparticles that did not correspond to identified cellularstructures, i.e., axon terminals or dendrites. Wheneverpossible, the more lightly labeled structures were con-firmed by detection of gold-silver particles in at least twoserial sections. A profile containing a small number of goldparticles (e.g., two gold particles) that was unlabeled inadjacent thin sections was designated as lacking detect-able immunoreactivity. Silver-intensification times duringthe histological processing were monitored to provideminimal background labeling in the neuropil. Therefore,as observed in low-magnification electron micrographs,cellular compartments containing at least three to fourgold particles compared with the neuropil were consideredto be immunoreactive.

RESULTS

By light microscopy, dOR immunoreactivity was distrib-uted moderately in the LC with the immunoperoxidaselabeling method (not shown). At higher magnification, theperoxidase reaction product for dOR immunoreactivityshowed a punctate distribution within the LC region, withno immunocytochemical labeling apparent in LC somata.Immunogold-silver labeling for M-ENK showed a promi-nent distribution in the LC, as reported previously (VanBockstaele et al., 1995), and immunolabeling for M-ENKwas restricted primarily to varicose processes. In dual-labeled sections of tissue and by using light microscopy,both markers, which appear similar in color, were difficultto clearly differentiate.

Ultrastructural localization of dOR

By electron microscopy, immunoperoxidase labeling fordOR was distributed along punctate patches of the plasmamembrane of axon terminals as well as on the membranesof large dense core vesicles (Figs. 1–3). Axon terminalsconstituted 80% (n 5 160) of the total population of dOR-labeled cellular profiles (n 5 200) examined in the presentstudy. The remaining profiles (n 5 40) consisted of den-drites, unmyelinated axons, and astrocytic processes (seebelow).

The dOR-labeled axon terminals contained small clearas well as large dense core vesicles. Peroxidase immunola-beling was often associated with large dense core vesicles(Figs. 1, 2–C, 3). At times, the dense core vesicles had clearcentral lumens with peroxidase immunoreactivity associ-ated primarily with the vesicular membrane (Fig. 1B).These were clearly disctinct from unlabeled dense core

dOR AND ENK IN LC 577

vesicles (udcv; Fig. 1B). These peroxidase-labeled densecore vesicles were often not located at the synaptic special-ization but were often identified along portions of theaxoplasm distal to the active zone (Figs. 1, 2–C, 3). Inaddition to dOR-labeled dense core vesicles, axon termi-nals also contained numerous small synaptic vesicles(Figs. 1–4) as well as unlabeled dense core vesicles (Figs.1B, 3C). Terminals containing dOR immunoreactivity weresometimes apposed by astrocytic processes along someportion of their plasma membrane (Figs. 1, 2A,C, 3A).Furthermore, immunolabeled axon terminals were alsoapposed to or formed synapses with unlabeled dendrites.

Synaptic specializations formed by dOR-labeled axonterminals included both type I (asymmetric) and type II(symmetric) synapses. The dOR-labeled terminals oftensynapsed with unlabeled dendrites. The dendrites re-ceived synaptic input from other unlabeled terminals(Figs. 1A, 2A, 4A,C). Of the axon terminals that containeddOR immunolabeling (n 5 160), 65% (n 5 104) formed

synaptic contacts with unlabeled dendrites. The remain-der did not form discernible synaptic specializations in thesections examined. Of the dOR-labeled axon terminalsthat formed synaptic contacts with unlabeled dendrites,38% formed asymmetric-type synaptic contacts, and 9%formed symmetric-type contacts, whereas the remainderdid not form recognizable synaptic specializations in theplane of section examined. These axon terminals con-tained heterogeneous types of synaptic vesicles, i.e., smallclear and large dense core vesicles (Figs. 2, 4A), andformed asymmetric (Fig. 4A) as well as symmetric contacts(Fig. 2) with unlabeled dendrites.

Some dOR-labeled axon terminalscontain M-ENK

Immunogold-silver labeling for M-ENK was localizedprimarily within axon terminals and unmyelinated axonsin the LC (Figs. 3, 4). Gold-silver labeling for M-ENK was

Fig. 1. Electron micrographs showing the vesicular content of axonterminals containing immunolabeling for the d-opioid receptor (dOR)in the nucleus locus coeruleus (LC). A: An axon terminal withnumerous small, synaptic vesicles also contains peroxidase labelingfor dOR in a large dense core vesicle (dcv). The axon terminal forms asynaptic contact (thick curved arrow) with an unlabeled dendrite (uD).The axon terminal is enveloped by an astrocytic process (asterisks).The uD receives convergent input from two other unlabeled terminals(ut). B: An axon terminal containing numerous small, clear vesicles aswell as several large dense core vesicles (dcv) forms an asymmetric

synaptic contact (thick curved arrow) with an unlabeled dendrite.Accumulation of peroxidase reaction product can be seen along theplasma membrane of the dOR-labeled axon terminal (thick straightarrow). Some dense core vesicles also contain peroxidase labeling fordOR, whereas some appear to be unlabeled (udcv). The axon terminalas well as the postsynaptic dendrite are apposed by astrocytic pro-cesses (asterisks). Note a small unmyelienated axon (a) that containsperoxidase immunoreactivity for dOR in the neuropil. Scale bar 50.5 µm.

578 E.J. VAN BOCKSTAELE ET AL.

Fig. 2. Electron micrographs showing synaptic specializationsformed by axon terminals containing peroxidase immunoreactivity forthe dOR in the nucleus locus coeruleus (LC). A: An axon terminalcontaining peroxidase labeling for dOR along its plasma membrane(thick straight arrows) and within large dense core vesicles (dcv) formsan asymmetric synaptic contact (thick curved arrow) with a smallunlabeled dendrite (uD). The small uD also receives convergent inputfrom an unlabeled terminal (ut). The dOR-labeled axon terminal isenveloped by an astrocytic process (asterisks). B: An axon terminalcontaining peroxidase immunoreactivity along its plasma membrane(thick straight arrow) and within large dense core vesicles (dcv) formsan asymmetric synaptic contact (thick curved arrow) with a smallunlabeled dendrite (uD). Note two unlabeled terminals (ut) with

numerous small synaptic vesicles in the neuropil. C: An axon terminalcontaining numerous small synaptic vesicles as well as two dense corevesicles (dcv; one of which appears to contain peroxidase immunoreac-tivity for dOR) forms an asymmetric synaptic contact (thick curvedarrow) with a dendrite. An unlabeled terminal (ut) is separated fromthe dOR-labeled axon terminal by astrocytic processes (asterisks). Theut contains an unlabeled dense core vesicle (udcv). An axon terminalcontaining gold-silver labeling for enkephalin (ENK) can also be seenin the neuropil. D: An astrocytic process contains peroxidase immuno-reactivity for dOR (arrows). This astrocytic process is adjacent to anunlabeled terminal (ut). Scale bars 5 0.4 µm in A, B; 0.3 µm in C; 0.2µm in D.

Fig. 3. Electron micrographs showing the distribution of immu-noperoxidase labeling for dOR (DOR) in axon terminals and immuno-gold-silver labeling (arrowheads) for methionine-enkephalin (ENK) inthe nucleus locus coeruleus. A: An axon terminal containing gold-silver deposits (arrowheads) for ENK is largely separated from anaxon terminal containing peroxidase-labeled, large, dense core vesicles(dcv) for dOR by an astrocytic process. The dOR-labeled axon terminalis in direct contact with an unlabeled dendrite (uD). One smallunmyelinated axon (a) contains peroxidase immunoreactivity for dOR.The arrow points to an astrocytic process that contains peroxidaselabeling for dOR. B: A gold-silver labeled axon terminal (arrowheads)that contains some dense core vesicles (dcv) forms an asymmetricsynaptic contact (open curved arrow) with an unlabeled dendrite. The

unlabeled dendrite also receives convergent input (solid curved arrow)from an unlabeled terminal (ut). Note two axons (a) that containperoxidase immunoreactivity for dOR. There are also numerousunmyelinated axons (ua) in the neuropil that lack peroxidase immuno-reactivity for dOR. An axon terminal containing a peroxidase-labeleddense core vesicle for dOR lies a short distance away from theENK-labeled axon terminal. C: A gold-silver-labeled ENK axon termi-nal (arrowheads) containing a dense core vesicle (dcv) is located ashort distance away from an axon terminal containing several densecore vesicles with peroxidase labeling for dOR. Two dense core vesiclesclearly contain peroxidase immunoreactivity for dOR, whereas someothers appear unlabeled (udcv). Scale bars 5 0.6 µm in A, 0.5 µm in B,0.4 µm in C.

Fig. 4. Electron micrographs showing the colocalization of peroxi-dase labeling for dOR (DOR) and immunogold-silver labeling for theopioid peptide enkephalin (ENK). A: An axon terminal containinggold-silver deposits for ENK as well as peroxidase immunoreactivityfor dOR (thick arrow) forms an asymmetric synaptic contact with anunlabeled dendrite (uD). The uD also receives convergent input froman unlabeled terminal (ut) containing several unlabeled large densecore vesicles (udcv). An astrocytic process (asterisks) is adjacent to thedOR-labeled axon terminal, the uD, and the ut. A process also containsperoxidase immunolabeling for dOR in the neuropil. B: An axon

terminal containing peroxidase labeling for dOR (large arrow) alsocontains gold-silver deposits for ENK. A small unmelinated axon (a)contains peroxidase labeling for dOR. An axon terminal containinggold-silver deposits for ENK can be seen at the bottom of themicrograph. C: An axon terminal containing both peroxidase labelingfor dOR (straight arrows) and gold-silver deposits for ENK forms anasymmetric type contact (curved arrow) with an unlabeled dendrite(uD). The uD also receives convergent from an unlabeled terminal (ut).The dual-labeled terminal and the uD are apposed by an astrocyticprocess (asterisks). Scale bars 5 0.5 µm in A, 0.7 µm in B, 0.4 µm in C.

preferentially associated with portions of the axoplasmcontaining large dense core vesicles. The dense core vesicleswere also commonly located at a distance from the synap-tic specialization.

Some dOR-labeled axon terminals contained M-ENKimmunoreactivity. Of the axon terminals that containeddOR immunoreactivity (n 5 160), 16% (n 5 27) containedimmunolabeling for M-ENK, and the remainder lackeddetectable levels of M-ENK.

dOR immunoreactivity in dendrites,unmyelinated axons and astrocytic processes

The dOR-labeled postsynaptic dendrites (Fig. 5) weremedium in size, ranging from 0.5 µm to 2.0 µm incross-sectional diameter (mean, 0.7 µm 6 0.2 µm). Othersmaller processes (,0.3 µm in cross-sectional diameter)also contained dOR immunoreactivity and usually con-tained few cytoplasmic organelles. Peroxidase immunore-activity appeared clumpy in dendrites and was not diffusewithin the cytoplasmic compartments.At times, the peroxi-dase labeling was close to the synaptic specializationformed by unlabeled terminals (Fig. 5C), or it was locatedaway from the synapse (Fig. 5B). The axon terminals inmost cases, however, contained unlabeled large dense corevesicles, suggesting that the terminals may contain aneuropeptide; however, these usually lacked immunolabel-ing for M-ENK.

Peroxidase labeling for dOR was also identified in astro-cytic processes (Figs. 4D, 5A,B) and unmyelinated axons(Fig. 5A,B). Portions of the neuropil containing dOR immu-noreactivity in astrocytic processes and unmyelinatedaxons sometimes also contained axon terminals withM-ENK, but this was not always the case (Fig. 5). How-ever, they were in proximity to unlabeled axon terminalscontaining unlabeled dense core vesicles (Fig. 5B).

DISCUSSION

The results of this study provide ultrastructural evi-dence that in the LC dOR immunoreactivity is localizedmainly to plasma membranes and to large dense corevesicles within axon terminals, some of which containimmunolabeling for M-ENK. The dOR-labeled dense corevesicles were located primarily near the perimeter of axonterminals. These data suggest that dOR functions topresynaptically modulate neurotransmitter release and inautoregulation of ENK release. The observed separationbetween dOR-labeled axons and many other ENK-contain-ing terminals suggests that the opioid peptide M-ENKmay be released by exocytosis and diffuse within theextracellular space to reach dOR-receptive sites located onnearby axon terminals. In addition, the occasional detec-tion of dOR in dendrites suggests a more minor involve-ment of dOR in postsynaptic effects that are associatedprimarily with the µOR. dOR may also produce changes inglial function, as suggested by localization to astrocytes.

Methodological considerations

Specificity of antiserum. By immunodot-blot analy-sis, the guinea pig antiserum raised against the dOR, p34,used at a concentration of 1:2,000, specifically recognizedthe immunizing dOR peptide p34 with a threshold of 8 ngbut did not cross-react appreciably with other nonimmuniz-ing peptides from adjacent positions on the dOR or withpeptides from segments of the cloned µORs or kORs

(Cheng et al., 1995). The antibody raised against dOR, p34,corresponds to amino acids 34–47, an extracellular frag-ment within the N-terminus. The antibody also showed adistribution in the LC and other regions that is similar tothe autoradiographic localization of dOR ligands (Atwehand Kuhar, 1977). The M-ENK antiserum principallyrecognized the parent peptide (Cheng et al., 1995), al-though some cross-reactivity was observed with the dynor-phin peptide. Because dynorphin peptides are prominentlydistributed within the LC, it is possible that the antiserumdirected against M-ENK used here may have partiallyrecognized some dynorphin profiles.

Data analysis. The quantitative approach used in thepresent study has been discussed previously (Van Bock-staele and Pickel, 1993; Van Bockstaele et al., 1994). Toensure the reproducibility of the quantitative evaluation ofthe types of junctions formed by immunoreactive processesand/or their frequency of association with other labeled orunlabeled cellular constituents, our analysis was re-stricted to sections collected from the surface of the tissue,which enabled both markers to be detected in all sectionsused for analysis (Chan et al., 1990).

Advantages and limitations of method. The subcel-lular distribution of dOR-like immunoreactivity in the LCin the present study is in agreement with previous studiesthat used autoradiography to localize dOR ligands in thisregion (Atweh and Kuhar, 1977; Tempel and Zukin, 1987;Arvidsson et al., 1995). The methodology employed here,however, has several advantages over receptor autoradiog-raphy, because it can indicate cellular sites for receptoractivation. For example, determining whether dOR isdistributed within neurons that are pre- or postsynaptic toopioid-containing afferents can best be addresed by usingultrastructural techniques. In the present analysis, werestricted our study to portions of the neuropil known to beenriched in noradrenergic perikarya and dendrites (Swan-son, 1976; Grzanna and Molliver, 1980).

However, our study does not establish unequivocallythat the dendrites that are postsynaptic to afferent termi-nals containing dOR are noradrenergic (Shipley, 1996).This would require triple-labeling procedures, which, atthe present time, are not feasible at the ultrastructurallevel. The single-section analysis used in the present studymay have also underestimated the relative frequencies ofassociations between M-ENK- and dOR-containing pro-files. The dOR is present is relatively low abundance and isalso extremely labile, being highly sensitive to detergentsthat increase penetration of antisera (Loh and Smith,1990). The use of acrolein as fixative and the lack of TritonX-100 used in these experiments most likely increased theefficacy of our immunolabeling method. Acrolein has beenshown previously to be advantageous for neuropeptidesand peptide fragments of G-protein-coupled receptors (Aokiand Pickel, 1994; Sesack et al., 1994). However, the caveatremains that our numbers do not reflect the true percent-age of colocalization between dOR and M-ENK.

Presynaptic distribution of dORimmunoreactivity

Ultrastructural analysis indicated that dOR was com-monly identified within axon terminals that containedheterogeneous types of synaptic vesicles in the LC. Withinthe axon terminals, immunolabeling was associated withthe plasma membrane of axon terminals as well with largedense core vesicles. The labeled dense core vesicles were

582 E.J. VAN BOCKSTAELE ET AL.

Fig. 5. Electron micrographs showing peroxidase labeling for dORwithin dendrites, unmyelinated axons, and glial processes in thenucleus locus coeruleus (LC). A: A dendrite containing peroxidaselabeling for dOR (open curved thick arrow) is located near an axonterminal containing unlabeled dense core vesicles (udcv). Note anunmyelinated axon (a) as well as an astrocytic process (solid straightthick arrow) that contain peroxidase labeling for dOR in the neuropil.B: A dendrite containing dOR (open curved arrow) is apposed by anunlabeled terminal containing several unlabeled dense core vesicles

(udcv). An intensely peroxidase-labeled unmyelinated axon (a) canalso be seen in the neuropil. Solid straight arrows point to anastrocytic process containing peroxidase labeling for dOR. C: A den-drite containing dOR (curved open arrow) is apposed by an unlabeledterminal (ut) containing several unlabeled dense core vesicles (udcv).Note that a gold-silver-labeled ENK axon with small vesicles can beseen in the neuropil. The ENK-labeled axon is enveloped by anastrocytic process (asterisks) that largely separates it from an unla-beled dendrite (uD). Scale bars 5 0.5 µm in A, C; 0.4 µm in B.

not commonly associated with the synaptic junction, butthey were often located on the perimeter of the axoplasm.The vesicular localization of dOR immunoreactivity raisesthe possibility that, in unstimulated axons, the dOR isprincipally retained in intracellular vesicular compart-ments and becomes incorporated into the membrane dur-ing exocytotic release of transmitters or peptide modula-tors, as suggested by Cheng et al. (1995) for laminae I andII of the dorsal horn in the cervical spinal cord. Incorpora-tion of dORs from vesicular membranes on axonal plasmamembranes may provide a means for further regulatingtransmitter release from the presynaptic terminal. Thelocalization of dOR to dense core vesicles also suggests thatthese organelles may be involved in transport of thereceptor from the soma to the axon terminal (Laduron andCastel, 1990). Localization of dOR to dense core vesiclessuggests that the receptor may participate in exocytoticrelease of transmitters from the vesicles. All opioid recep-tors are thought to be coupled to G-proteins, and they areall thought to inhibit adenylyl cyclase (Childers, 1991).The G-proteins, initially believed to be present exclusivelyin the plasma membrane, have recently been found to beassociated with intracellular membrane compartments(Aronin and Di Figlia, 1992). Furthermore, G-proteinshave been localized to dense core vesicles in bovine adrenalmedulla (Ahnert-Hilger et al., 1994). Therefore, activationof dOR may influence release of neurotransmitter fromvesicular compartments.

The localization of dOR to axon terminals suggests thatthis receptor is located on afferent terminals that impingeon the LC. There are several nuclei that may provideafferent input to the LC (Luppi et al., 1995). These includethe nucleus paragigantocellularis and the nucleus preposi-tus hypoglossi in the rostral medulla (Aston-Jones et al.,1991; Drolet et al., 1992; Ennis et al., 1992), the periaque-ductal gray (Ennis et al., 1991), the Kolliker Fuse (Luppi etal., 1995), the central nucleus of the amygdala (VanBockstaele et al., 1996c), and the medial prefrontal cortex(Chiang et al., 1987), among others. The morphology of thesynaptic specialization suggests that the dOR-labeled axonterminals may be colocalized with excitatory or inhibitoryneurotransmitters (Carlin et al., 1980). Future studies arerequired to determine which afferents possess dOR immu-noreactivity.

The dOR-labeled axon terminals were commonly ap-posed by astrocytic processes and may indicate a cellularsubstrate for signaling between neurons and astrocytes.Glial processes may facilitate diffusion of the transmitterreleased from the axon terminal in the neuropil and, thus,affect postsynaptic modulation of the target (Pickel et al.,1995). In addition, however, the astrocytes could produceENK, because some astrocytic processes have been shownto contain peroxidase immunoreactivity for leucine5-ENK(Stiene-Martin and Hauser 1993; Stiene-Martin et al.,1993). b-adrenergic receptors stimulate the production ofpreproenkephalin in astrocytic cultures. This evidencesuggests a potentially novel form of communication inwhich noradrenergic release from dendrites or afferents tothe LC may stimulate the production of ENK that isreleased to activate dOR sites in the LC.

Relationships with M-ENK

The endogenous opioid peptide, M-ENK, was foundoccasionally in axon terminals containing dOR in the LC,suggesting that the dOR may function as an autoreceptor.

When the dOR and M-ENK were identified in the sameaxon terminal, small clear as well as large dense corevesicles were often contained within the cytoplasm of theterminal. Immunoreactivities for M-ENK and dOR wereoften localized to portions of the plasmalemma distal to thezone of synaptic junction, suggesting that the peptide maybe released from sites other than at the active zone, asreported for other brain regions (Zhu et al., 1986; Cheng etal., 1995). The synaptic specialization was often of theasymmetric variety, suggesting that the terminal maycontain an excitatory transmitter, such as glutamate(Gray, 1959; Peters et al., 1991).

Dendritic associations of dOR

In the region sampled for ultrastructural analysis, den-drites did not usually contain M-ENK, although the opioidpeptide has been shown to be present in LC cell bodies inother species (Charnay et al., 1982). Some dendrites,however, did contain dOR immunoreactivity. Such dOR-labeled dendrites received synaptic contacts from axonterminals that lacked detectable M-ENK immunolabeling.The unlabeled terminals sometimes contained heterog-enous types of synaptic vesicles, such as small clear andlarge dense core vesicles, suggesting that they may colocal-ize a neuropeptide.Apossible candidate for the neurotrans-mitter in these unlabeled axon terminals is leucine5(L)-enkephalin, which is also known to be an endogenousligand of dOR. Future studies are required to determinewhether L-ENK is found in a different population of axonterminals contacting dOR-labeled dendrites.

Functional implications

The results of this study indicate several cellular sites atwhich dOR may contribute to the modulatory actions ofneurotransmitters and endogenous opioid peptides. Thedata also indicate a sharp contrast in the subcellulardistribution of two different opioid receptors. We havepreviously shown that µOR immunoreactivity was oftenidentified along the plasma membranes of perikarya anddendrites containing or lacking the catecholamine synthe-sizing enzyme, tyrosine hydroxylase (Van Bockstaele et al.,1996a). The presence of µOR in perikarya and dendritesand its apparent infrequent distribution in axons and axonterminals suggest that this receptor may function primar-ily in a postsynaptic manner. µ-Opioid receptor agonistshave been reported to inhibit the spontaneous activity ofLC neurons via a pertussis toxin-sensitive G protein(North and Williams, 1985; Aghajanian and Wang, 1986;North et al., 1987), to inhibit adenylate cyclase activity(Duman et al., 1988; Beitner et al., 1989), and to decreasec-AMP-dependent protein phosphorylation (Guitart andNestler, 1989, 1993) in neurons of the LC. Our previousstudy of µOR localization supports a cellular substrate fordirect postsynaptic effects of ligands binding to the µOR.

The results of the present study, however, indicate thatthe dOR is located primarily presynaptically. This differ-ence in receptor distribution may be important for physi-ological effects exerted by opioid peptides in this brainregion. It is possible that activation of µOR and dOR mayoccur under different physiological situations or that theycause different responses to administration of exogenousopiates. In addition, the endogenous opioid peptides M-ENK and L-ENK act nonspecificically at both µOR anddOr, suggesting that these peptides may differentiallyaffect opioid receptors in the LC. Endogenous opioid

584 E.J. VAN BOCKSTAELE ET AL.

release in the LC has been shown to occur during stress.Abercrombie and Jacobs (1988) found that naloxone poten-tiated the increase in LC activity in response to stress, but,in a resting state, naloxone did not alter LC activity. Thesedata suggest that endogenous M-ENK- or L-ENK-contain-ing afferents may be activated by different stimuli toinfluence µORs and dORs in the LC. Each opioid peptidemay derive from different afferent nuclei, and potentialdifferences in opioid circuitry influencing the LC are beingpresently investigated.

ACKNOWLEDGMENTS

This work was supported by R29 DA09082 and RO3DA10450 from the National Institute on Drug Abuse;MH40008 from the National Institute on Mental Health,National Institutes of Health (NIH) and an EstablishedInvestigator Award from the American Heart Associationto E.J.V.B; and MH00078 to V.M.P.

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