THE JOURNAL OF COMPARATIVE NEUROLOGY 37665-74 (1996)

Mu-Opioid Receptor Is Located on the Plasma Membrane of Dendrites That

Receive Asymmetric Synapses From Axon Terminals Containing Leucine-Enkephalin

in the Rat Nucleus Locus Coeruleus

E.J. VAN BOCKSTAELE, E.E.O. COLAGO, A. MORIWAKI, AND G.R. UHL Department of Neurology and Neuroscience, Cornell University Medical College, New York,

New York 10021 (E.J.V.B., E.E.O.C.); NIDA-Division of Intramural Research Program, Addiction Research Center, Baltimore, Maryland 21224 (A.M., G.R.U.)

ABSTRACT We have recently shown, by using immunoelectron microscopy, that the mu-opioid

receptor (FOR) is prominently distributed within noradrenergic perikarya and dendrites of the nucleus locus coeruleus (LC), many of which receive excitatory-type (i.e., asymmetric) synaptic contacts from unlabeled axon terminals. To characterize further the neurotransmitter present in these afferent terminals, we examined in the present study the ultrastructural localization of an antipeptide sequence unique to the FOR in sections that were also dually labeled for the opioid peptide leucine-enkephalin (L-ENK). Immunogold-silver labeling for FOR was localized to extrasynaptic portions of the plasma membranes of perikarya and dendrites. The FOR- labeled dendrites were usually postsynaptic to axon terminals containing heterogeneous types of synaptic vesicles and forming asymmetric synaptic specializations characteristic of excitatory- type synapses. The majority of these were immunolabeled for the endogenous opioid peptide L-ENK. Some FOR-labeled dendrites received synaptic contacts from unlabeled axon terminals in fields containing L-ENK immunoreactivity. In such cases, the FOR-labeled dendrites were in proximity to L-ENK axon terminals that contained intense peroxidase labeling within large dense core vesicles along the perimeter of the axoplasm. These results indicate that L-ENK may be released by exocytosis from the dense core vesicles and diffuse within the extracellular space to reach FOR sites on the postsynaptic dendrite or dendrites of other neighboring neurons. The present study also reveals that unlabeled terminals apposed to FOR-labeled dendrites may contain other opioid peptides, such as methionine-enkephalin. These data demonstrate several sites where endogenous opioid peptides may interact with FOR receptive sites in the LC and may provide an anatomical substrate for the LC’s involvement in mechanisms of opiate dependence and withdrawal.

Indexing terms: norepinephrine, drug abuse, enkephalin, opiates, morphine

D 1996 wiey-Liss, Inc.

We have recently demonstrated that the mu-opioid recep- tor (FOR) is prominently distributed within norepinephrine- containing neurons in the locus coeruleus (LC) in the rostra1 dorsal pons (Van Bockstaele et al., 1996b). Further- more, our study showed that unlabeled axon terminals forming primarily asymmetric synaptic junctions character- istic of excitatory neurotransmitters formed synapses with dendrites containing FOR. These findings were compelling because of substantial evidence showing that the physical symptoms observed upon withdrawal from opiates may be mediated by enhanced excitatory amino acid transmission within the LC (Rasmussen et al., 1990; Akaoka and Aston-

Jones, 1991; Aghajanian et al., 1994; Zhang et al., 1994). Discharge activity of LC neurons is significantly elevated during naloxone-precipitated withdrawal (Rasmussen et al., 1990). Microdialysis studies have indicated an increase in the efflux of excitatory amino acids in the LC following naltrexone-precipitated withdrawal (Aghajanian et al., 1994;

Accepted July 11, 1996. Address reprint requests to Elisabeth J. Van Bockstaele Ph.D., Depart-

ment of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, 1020 Locust Street, Rm 244, Philadelphia, PA 19107.

o 1996 WILEY-LISS, INC.

66 E.J. VAN BOCKSTAELE ET AL.

Zhang et al., 1994). Injections of the excitatory amino acid antagonist kynurenic acid (Rasmussen and Aghajanian, 1989) intracerebroventricularly or directly into the LC (Akaoka and Aston-Jones, 1991) have been shown to attenu- ate significantly the activation of LC cells induced by naloxone-precipitated withdrawal. Lesions of the nucleus paragigantocellularis (PGi), the major excitatory amino acid pathway to the LC, attenuate the hyperactivity seen following precipitated opiate withdrawal (Rasmussen and Aghajanian, 1989). These lines of evidence strongly support the role of excitatory afFerents from the PGi containing primarily glutamate in the mediation of withdrawal- induced hyperactivity in LC (Rasmussen et al., 1990).

However, the neurotransmitter within the unlabeled axon terminals apposed to FOR-labeled perikarya and dendrites identified in our previous study (Van Bockstaele et al., 1996b) is unknown. Possible candidates include the opioid peptides leucine- and methionine-enkephalin, which are known to be endogenous ligands of the FOR (Bird and Kuhar, 1977). However, the asymmetry of the synaptic specialization observed in our previous study (Van Bocks- taele et al., 1996b) suggests that the transmitter could be an excitatory amino acid (Peters et al., 1991). Interestingly, opioid-and glutamate-containing neurons in the PGi pro- vide significant afferent input to the LC (Ennis and Aston- Jones, 1986; Drolet and Aston-Jones, 1991; Drolet et al., 1992). Thus, we examined, in the same section of tissue, the ultrastructural immunocytochemical localization of an an- tipeptide antibody generated against a specific peptide sequence uniquely present in pOR (Surratt et al., 1994) with the detection of an antibody to the opioid peptide leucine- enkephalin (L-ENK). These results provide the first ultrastruc- turd demonstration that FOR is extensively localized to extra- synaptic sites along the plasma membrane of dendrites that receive excitatory-type synapses from L-ENK-containing axon terminals in the LC. These data also suggest potential sites of recephr activation on plasmalemmal sites of dendrites that receive afferents from unlabeled axon terminals.

MATERIALS AND METHODS Tissue preparation

Four adult male Sprague Dawley rats (Taconic Farms; 200-250 g) were used in this study. Animals were deeply anesthetized with sodium pentobarbital and perfused trans- cardially through the ascending aorta with 50 ml of 3.8% acrolein and 200 ml of 2% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). Immediately following perfusion-fixation, the brains were removed, cut into 1-3 mm coronal slices, and placed in the same fixative for an additional 30 minutes. Forty-micrometer-thick sections were cut through the rostrocaudal extent of the LC using a Vibratome and collected into 0.1 M PB.

Antisera specificity The characterization and specificity of the rabbit antise-

rum against the mu-opioid receptor and the mouse anti- body against L-ENK have been described previously (Sur- ratt et d., 1994; Van Bockstaele et al., 1996b). The rabbit polyclonal antiserum was raised against a glutaraldehyde conjugate of the C-terminal 18 amino acids of the rat pOR receptor and keyhole limpet hemocyanin. Immunolabeling was selectively adsorbed with the appropriate peptide, with concentrations of 1 and 10 pg/ml (Van Bockstaele et al., 199613). Immunodot-blot analysis (Cheng et al., 1996) was

also used to show specificity of the rabbit antiserum against the pOR peptide. The L-ENK antibody was shown to recognize primarily L-ENK and not methionine-enkephalin or dynorphin A (Van Bockstaele et al., 1996a). The immuno- reaction product was shown to be abolished in tissue sections through the brainstem by preadsorption of the L-ENK antibody with high concentrations of L-ENK (Svin- gos et al., 1996; Van Bockstaele et al., 1996b). To evaluate the possible recognition of the primary rabbit antiserum by secondaries against mouse IgGs in the dual-labeling experi- ments, some sections were processed for dual labeling with omission of the mouse antiserum.

Immunocytochemical labeling The Vibratome sections were placed for 30 minutes in 1%

sodium borohydride in 0.1 M PB to remove reactive alde- hydes (Leranth and Pickel, 1989). Sections were then rinsed extensively in 0.1 M Tris-buffered saline (TBS) and incubated for 30 minutes in 0.5% bovine serum albumin (BSA) in 0.1 M TBS prior to the primary antibody incuba- tion. Tissue sections containing the LC were incubated in a cocktail of rabbit anti-pOR antibody (1:10,000; Surratt et al., 1994) and a mouse monoclonal anti-L-ENK antibody (1:100, Sera Laboratories). Sections were treated with 0.3% Triton X-100 for light microscopy but lacked Triton X-100 for electron microscopy.

Methods for dual immunocytochemical labeling have been previously described (Chan et al., 1990; Van Bocks- taele et al., 1994). The FOR was immunolabeled by using the immunogold-silver method, and L-ENK was identified by using the immunoperoxidase labeling method for ultra- structural observations. Tissue sections were incubated in the primary antibody 15-18 hours at room temperature. They were then rinsed three times in 0.1 M TBS and incubated at room temperature for 30 minutes in biotinyl- ated goat anti-mouse (1:400; Vector Laboratories) for immu- noperoxidase labeling of L-ENK.

For peroxidase labeling of L-ENK, sections were then incubated in avidin-biotin complex for 30 minutes (Vector Laboratories; Hsu et al., 1981). For all incubations and washes, sections were continuously agitated by using a Thomas rotator. L-ENK was then visualized by a 4 minute reaction in 22 mg of 3-3’-diaminobenzidine (Aldrich) and 10 p1 of 30% hydrogen peroxide in 100 ml of 0.1 M TBS. For pOR immunocytochemistry, these sections were subse- quently rinsed in 0.01 M phosphate-buffered saline (PBS) and incubated in a solution of 0.01 M PBS containing 0.1% gelatin and 0.8% BSA for 30 minutes. Sections were then incubated in a goat anti-rabbit IgG conjugated to 1 nm gold particles (Amersham Corp.) for 2 hours at room tempera- ture. These were rinsed in 0.01 M PBS containing the same concentrations of gelatin and BSA described above and subsequently rinsed with 0.01 M PBS. Sections were then incubated in 1.25% glutaraldehyde in 0.01 M PBS for 10 minutes, followed by a wash in 0.01 M PBS and then in 0.2 M sodium citrate buffer (pH 7.4). Silver intensification of the gold particles was achieved by using a silver enhance- ment kit (Amersham Corp.). The optimal silver enhancement times were determined empirically for each experiment and ranged from 7 to 8 min for electron microscopy and from 11 to 12 min 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, and coverslipped in DPX neutral mounting medium (Aldrich). For electron microscopy, sections were rinsed in 0.1 M PBS

pOR AND ENK IN LC

and incubated in 2% osmium tetroxide in 0.1 M PB for 1 hour, washed in 0.1 M PB, dehydrated, and flat embedded in Epon 812 (Leranth and Pickel, 1989). Thin sections of approximately 55-65 nm were cut from the outer surface of the tissue with a diamond knife (Diatome) by using an RMC ultramicrotome. These were collected on grids and counter- stained with uranyl acetate and Reynolds lead citrate.

67

Data analysis Thin sections of tissue prepared for electron microscopy

were selected immediately adjacent to the fourth ventricle corresponding to Plate 50 of the rat brain atlas of Swanson (1992). Analysis was performed on thin sections collected sufficiently close to the outer surface of the tissue to permit detection of both FOR and L-ENK immunoreactivities. The classification of identified cellular elements was based on the work of Peters et al. (1991). Structures were defined as being proximal dendrites if they contained endoplasmic reticulum and were larger than 0.7 pm in diameter. Axon terminals were distinguished from unmyelinated axons based on their content of synaptic vesicles and diameter greater than 0.1 Fm. A terminal was considered to be synaptic when it showed a junctional complex, a restricted zone of parallel membrane apposition with slight enlarge- ment of the intercellular space, and/or associated postsyn- aptic thickening. Asymmetric synapses were identified by the presence of thick postsynaptic densities (Gray’s type I; Gray, 1959); symmetric synapses, on the other hand, had thin densities (Gray’s type 11; Gray, 1959) both pre- and postsynaptically. Nonsynaptic contacts, or appositions, were defined by closely spaced parallel plasma membranes of immunoreactive axons and other axon terminals or den- drites. These lacked recognizable specializations and were not separated by glial processes.

Tissues from animals with the best immunocytochemical labeling and preservation of ultrastructural morphology were included in the analysis. At least ten grids containing five to ten thin sections each were collected from the surface of three or more plastic-embedded sections containing the LC from each animal. Photographs were taken only when both markers were clearly in the same neuropil in fields magnified x 10,000.

RESULTS With light microscopy, FOR immunoreactivity was promi-

nently localized to the LC using either immunoperoxidase (Fig. lA,B) and immunogold-silver methods (not shown). At higher magnification, the peroxidase reaction product for pOR immunoreactivity showed a punctate distribution within LC perikarya and dendrites (Fig. 1B) with no immunocytochemical labeling apparent in their nuclei (Fig. 1B). Immunoperoxidase (not shown) and immunogold- silver labeling for L-ENK (Fig. 1C,D) also showed a promi- nent distribution in the LC, as was previously reported (Van Bockstaele et al., 1995, where immunolabeling for L-ENK was restricted primarily to varicose processes (Fig. lC,D). In dually labeled sections of tissue, both markers, which appear similar in color, were difficult to differentiate clearly (Fig. 1C) when the pOR was labeled with the gold-silver marker and L-ENK was labeled with peroxidase. Therefore, for light microscopy tissue sections, the markers were reversed. At high magnification (Fig. lD), peroxidase immunoreactivity could be seen in cell bodies and immuno- gold-silver labeling for L-ENK could be seen in varicose

processes and puncta, which overlapped FOR immunoreac- tivity in the LC.

Differential distribution of pOR and L-ENK immunoreactivities

With electron microscopy, immunogold-silver labeling for pOR was often localized to perikarya, to dendrites (Figs. 2, 31, and, occasionally, to unmyelinated axons and glial processes in the LC. Spurious gold-silver labeling for pOR that was not associated with cellular membranes was negligible. Gold-silver labeling for pOR was preferentially associated with the cytoplasmic surfaces of extrasynaptic plasma membranes of dendrites and perikarya (Figs. 2-4). The gold-silver particles were extensively localized along extrasynaptic (Figs. 2, 3) portions of the dendritic mem- branes. Extrasynaptic labeling was defined by the presence of gold-silver deposits along any portion of the plasma membrane of dendrites (Figs. 2,3), whether or not synaptic input was seen within the sections examined. Dendrites containing FOR immunoreactivity along extrasynaptic por- tions of their plasma membranes were often apposed by astrocytic processes that usually lacked FOR-labeling (Figs. 3, 4). Occasionally, gold-silver particles were even more closely associated with the active zone of dendrites receiv- ing synaptic contacts from unlabeled terminals. However, these were still usually located somewhat lateral to the postsynaptic specialization. Gold-silver particles were also occasionally distributed within the cytoplasm of dendrites. These were usually seen near saccules of smooth endoplas- mic reticulum.

Peroxidase labeling for L-ENK was restricted to unmyelin- ated axons and axon terminals (Figs. 2,3). Some astrocytic processes also contained immunolabeling for L-ENK (Fig. 4A), and these were often associated with intensely labeled L-ENK axon terminals. Axon terminals containing L-ENK immunoreactivity contained heterogeneous types of synap- tic vesicles. These included small clear vesicles as well as intensely peroxidase labeled large dense core vesicles (Figs. 2-4). The large dense core vesicles were found along the perimeter of the L-ENK-labeled axon terminal distal to the active zone of the synapse. Glial processes were often apposed to the L-ENK-labeled axon terminal along some portion of its plasma membrane (Figs. 2,3A, 4B). Interest- ingly, the dense core vesicles were located near portions of the axonal plasmalemma near astrocytic processes (Fig. 4B). The astrocytic processes directly intervened between neighboring dendrites (Fig. 2,4A).

pOR is localized to dendrites receiving asymmetric contacts from L-ENK terminals Dendrites constituted 95% (n = 285) and perikarya

constituted 5% (n = 15) of the total population of FOR- labeled neuronal profiles (n = 300) examined in the present study. The pOR-labeled recipient dendrites were medium in size, ranging from 0.5 to 2.0 p,m in cross-sectional diameter (mean 0.7 +- 0.2 Fm). Other, smaller processes (<0.3 pm in cross sectional diameter) also contained pOR-like immunoreactivity. These usually contained few cytoplasmic organelles and had ultrastructural features characteristic of dendritic spines.

Of the dendrites containing pOR immunoreactivity (n = 285), almost 60% (n = 171) were apposed by axon termi- nals. Of the axon terminals in direct contact with FOR- labeled dendrites (n = 1711, 40% (n = 68) contained peroxidase labeling for L-ENK. Of the L-ENK-labeled axon

68

Fig. 1. Light microscopy showing the cellular localization of the mu-opioid receptor (FOR) and its relation to leucine-enkephalin (L- ENK) in the nucleus locus coeruleus (LC). A In the dorsal pons at the level of Plate 52 in the rat brain atlas of Swanson (1992), dense peroxidase reaction product for pOR is seen immediately adjacent to the fourth ventricle in the LC (large arrow). Immunolabeled processes extend medially (small arrow) in a zone known to contain dendrites of the noradrenergic neurons of the LC. cb, cerebellum; scp, superior cerebellar peduncle. Double arrows point dorsally (D) and laterally (L). B A higher magnification photomicrograph from the same section as in A showing FOR immunoreactivity near the perimeter of perikarya whose cytoplasm and nuclei appear unlabeled. Note extensive dendrites

EJ. VAN BOCKSTAELE ET AL.

(d) that emanate from the densely labeled LC cell body LC-(cb) region. IV, fourth ventricle. C: Photomicrograph of a dually labeled section showing immunoperoxidase labeling of the pOR and gold-silver label- ing of L-ENK. A dense region of immunolabeling for L-ENK (arrows) can be seen immediately ventral to the superior cerebellar peduncle (scp). Asterisk denotes a blood vessel. D Higher magnification of the section shown in C depicting varicose processes containing gold-silver labeling for L-ENK (arrows). The asterisk denotes a blood vessel that can also be seen in C. Note that the nuclei of LC neurons are void of immunoreactivity. Scale bars = 250 p m in A, 100 in B, 75pm in C, 25 pm in D.

Fig. 2. Low-magnification electron micrograph showing differential immunolabeling of the mu-opioid receptor (FOR) and leucine- enkephalin (ENK) in the nucleus locus coeruleus (LC). A peroxidase- labeled axon terminal for ENK forms a synaptic contact (open arrow) with a dendrite containing a few gold-silver deposits (arrowheads) for pOR. Gold-silver deposits can be seen associated with the plasma membrane of dendrites (arrowheads) or within their cytoplasm (curved solid arrows). The ENK axon terminal is apposed to an astrocytic process (asterisks) that separates it from an unlabeled dendrite (uD). The postsynaptic target of the ENK terminal is separated from two dendrites containing gold-silver deposits for FOR by an astrocytic

process (asterisks). The more dorsally located dendrite is apposed to a myelinated axon (ma) and receives a synaptic contact (straight solid arrows) from an unlabeled terminal (ut). The larger dendrite contains a Golgi apparatus ( G ) and several mitochondria (m) and is separated from two unlabeled dendrites (uD) by astrocytic processes (asterisks). The uD on the left of the micrograph receives a synaptic contact from an unlabeled terminal (ut). The u t is enveloped by an astrocytic process that separates it from a densely gold-silver labeled (arrowheads) dendrite. mvb, multivesicular body; Ly, lysosome; er, endoplasmic reticulum; ma, myelinated axon. Scale bar = 2 km.

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Fig. 3. Plasmalemmal distribution of immunogold-silver labeling for mu-opioid receptor (pOR) in dendrites receiving synaptic contacts from axon terminals containing leucine-enkephalin (ENK) in the nucleus locus coeruleus. A: A dendrite containing gold-silver deposits (arrowheads) for +OR receives an asymmetric synaptic contact (open arrow) from an axon terminal containing peroxidase labeling for ENK. The ENK-labeled axon terminal contains numerous small clear vesicles as well as large dense core vesicles (dcv) and is apposed to an astrocytic process (asterisks). Note that numerous gold-silver particles can be seen to the left of the dendrite (arrowheads). The dendrite is also

EJ. VAN BOCKSTAELE ET AL.

apposed to a glial process and a vesicular-filled process (p). Another dendrite in the neuropil contains gold-silver deposits for FOR but lacks afferent input in this plane of section. A large unlabeled dendrite (uD) can also be identified in the neuropil. B: Aperoxidase labeled ENK axon terminal containing numerous dense core vesicles (dcv) forms an asymmetric type synaptic contact (open arrow) with a dendrite contain- ing gold-silver deposits (arrowheads) for pOR. Note prominent gold- silver labeling for FOR in dendrites not receiving any afferent input from the ENK axon terminal. Two unlabeled dendrites (uD) can also be seen in the neuropil. Scale bars = 0.4 pm in A, 0.7 pm in B.

Fig. 4. Electron micrographs showing gold-silver labeling (arrow- heads) for pOR on plasmalemmal sites distal to the active zone of L-ENK afferents and targeted by unlabeled terminals. A: A L-ENK- labeled axon terminal containing several dense core vesicles (dcv) forms an asymmetric synaptic contact (open arrow) with a large dendrite that lacks gold-silver labeling for FOR. The L-ENK axon terminal is apposed to an astrocytic process (solid arrow) that contains peroxidase labeling for L-ENK. An intensely gold-silver labeled dendrite is apposed to the

L-ENK terminal and receives a synaptic contact from an unlabeled terminal containing a dcv. B: A L-ENK-labeled axon terminal is apposed to an astrocytic process (asterisks) and a dendrite containing gold-silver deposits for FOR (arrowheads). A small and large dendrite contain intense gold-silver deposits for the pOR. The large dendrite contains a multivesicular body (mvb) and is enriched in endoplasmic reticulum (er). m, Mitochondria. Scale bars = 0.5 +m in A, 0.4 pm in B.

72 E.J. VAN BOCKSTAELE ET AL.

terminals forming synaptic contacts with pOR-labeled den- drites (n = 681, 55% (n = 37) formed asymmetric type synaptic contacts, 12% (n = 8) formed symmetric type contacts, and the remainder did not form recognizable synaptic specializations in the plane of section examined.

Unlabeled terminals form synaptic contacts with FOR-labeled dendrites

Some FOR-labeled dendrites received synaptic contacts from unlabeled terminals and lacked afferent input from L-ENK axon terminals, although L-ENK immunoreactivity was present in the neuropil in single sections examined. The unlabeled axon terminals contained heterogeneous types of synaptic vesicles, i.e., small clear and large dense core vesicles (Figs. 2,4A). The unlabeled terminals formed asymmetric (Fig. 4A) as well as symmetric contacts (Fig. 2) with the FOR-labeled dendrites.

DISCUSSION The results of this study provide ultrastructural evidence

that mu-opioid receptor (FOR) immunoreactivity is local- ized to the plasma membranes of dendrites that frequently receive asymmetric (excitatory-type) synapses from axon terminals containing leucine-enkephalin (L-ENK). The FOR labeling was, however, usually not detected at the postsyn- aptic junction but was localized to extrasynaptic sites on the plasma membrane. L-ENK immunoreactivity was most intensely localized to large dense core vesicles, which were often distributed on the perimeter of the axon terminal. The pOR immunoreactivity was also associated with plasma membranes of dendrites not targeted by the L-ENK affer- ents in some single sections analyzed. These data suggest that the opioid peptide may be released by exocytosis and that it diffuses within the extracellular space to reach pOR receptive sites located on the postsynaptic dendrite or dendrites of other neighboring neurons.

Methodological considerations Specificity of antiserum. The protein identified with

the FOR antiserum is most likely FOR based on 1) previous experiments showing the characterization of the polyclonal antiserum used in the present study (Surratt et al., 1994) 2) selective, adsorbable immunocytochemical labeling within various brain regions (Cheng et al., 1996; Svingos et al., 1996; Van Bockstaele et al., 1996b), and 3) immunodot-blot preparations made in our laboratory showing that the antibody recognizes amino acid sequences within pOR, but not sequences of delta or kappa receptors (Cheng et al., 1996). The antibody also shows a distribution in the LC and other regions that is similar to the autoradiographic localiza- tion of FOR ligands (Atweh and Kuhar, 1977). Most of the reaction product for pOR was localized to the cytoplasmic side of the plasma membranes of perikarya and dendrites. This localization is consistent with current models in which the pOR carboxyl terminus amino acid sequence is thought to be cytoplasmic (Wang et al., 1993). FOR immunoreactiv- ity was also associated with saccules of smooth endoplasmic reticulum within the cytoplasm. Such organelles have been shown to be involved in the intracellular transport of plasma membranes from the cell soma to dendrites and axon terminals (Rodriguez-Boulan and Powell, 1992).

The quantitative approach used in the present study has previously been discussed (Van Bocks- taele and Pickel, 1993; Van Bockstaele et al., 1994). To

Data analysis.

ensure the reproducibility of the quantitative evaluation of the types of junctions formed by immunoreactive processes andlor their frequency of association with other labeled or unlabeled cellular constituents, our analysis was restricted to sections collected from the surface of the tissue, which ensured that both markers were detectable in all sections used for analysis (Chan et al., 1990).

The intense distribution of pOR-like immunoreactivity in the LC in the present study is in agreement with previous studies that used autoradiog- raphy to localize pOR ligands in this region (Atweh and Kuhar, 1977) as well as with studies showing that mu- opioid mRNA is expressed by cells in the LC area (Mansour et al., 1995). The methodology employed here, however, has several advantages over receptor autoradiography (Atweh and Kuhar, 1977; Tempe1 and Zukin, 19871, insofar as it can indicate cellular sites for receptor activation. For example, determining whether FOR is distributed within neu- rons that are postsynaptic to opioid-containing afl'erents can best be addressed by using ultrastructural techniques.

Advantages of the method.

Distribution of FOR-like immunoreactivity The pOR immunoreactivity was identified predomi-

nantly along extrasynaptic portions of the plasma mem- branes of perikarya and dendrites and was not commonly associated with the synaptic junction. The largely extrasyn- aptic accumulation of reaction product for FOR immunore- activity most likely represents the functional subcellular distribution of the FOR protein due to the observed similar- ity in immunolabeling obtained using either immunogold- silver or peroxidase methodologies. The detection at extra- synaptic sites with both methods makes it unlikely that this distribution reflects only incomplete penetration to postsyn- aptic zones, as has been suggested for other receptors (Nusser et al., 1995). However, results from postembedding immunogold-silver analysis are necessary to establish un- equivocally that the receptor is not commonly located at the synaptic junction.

We have previously shown that FOR immunoreactivity was commonly identified along the plasma membranes of perikarya and dendrites containing or lacking the catechol- amine-synthesizing enzyme tyrosine hydroxylase (TH; Van Bockstaele et al., 1996b). The presence of pOR in perikarya and dendrites and its apparent infrequent distribution in axons and axon terminals suggested that this receptor may function primarily in a postsynaptic manner. In the present study, we restricted our analysis to portions of the neuropil known to be enriched in noradrenergic perikarya and dendrites. However, the present study does not unequivo- cally establish that the recipient dendrites of L-ENK affer- ent terminals are noradrenergic. This would require triple labeling procedures that, at the present time, are not feasible at the ultrastructural level.

Input from L-ENK axon terminals forming asymmetric synapses

Mu-opioid receptor agonists have been reported to inhibit the spontaneous activity of LC neurons via a pertussis toxin-sensitive G protein (North and Williams, 1985; Agha- janian and Wang, 1986; North et al., 1987). Acutely, opioid agonists also inhibit adenylate cyclase activity (Duman et al., 1988; Beitner et al., 1989) and decrease CAMP-dependent protein phosphorylation (Guitart and Nestler, 1989, 1993) in neurons of the LC. Acute opiate administration also decreases the turnover of norepinephrine (NE) in forebrain regions that

pOR AND ENK IN LC

receive aEerents from the LC (Arbilla and Langer, 1978). Interestingly, many of the L-ENK terminals forming synaptic contacts with FOR-labeled dendrites possessed junctions that were characteristic of excitatory transmission. Asymmetric synapses are believed to mediate excitation based largely on the detection of enriched populations of thickened postsynaptic densities in regions of the brain containing higher proportions of excitatory synapses (Carlin et al., 1980).

The presence of asymmetric synapses opposing plasma- lemma1 sites containing FOR immunoreactivity suggests sites where the released peptide may act postsynaptically, possibly through modulation of glutamate. Injections of kynurenic acid, a general antagonist of excitatory amino acids, into the LC significantly attenuated the activation of LC cells induced by naloxone-precipitated withdrawal (Ras- mussen et al., 1990). This suggested that an important part of the hyperactivity of LC neurons during opiate with- drawal may be mediated by an excitatory amino acid input (Akaoka and Aston-Jones, 1991).

The potential colocalization of endogenous opioid pep- tides and glutamate has yet to be unequivocally established in individual axon terminals of the LC but is strongly suggested by the present study and our previous study (Van Bockstaele et al., 1995). Interestingly, physiological studies have shown little evidence for opioid influences on LC neurons deriving from the PGi. This is in sharp contrast to the potent inhibitory effects induced by exogenously admin- istered opiates on LC neurons (see above). Differential release of the excitatory transmitter and the opioid peptide could potentially explain these discrepancies. Endogenous opioid release may occur only under conditions or stimulus frequencies that have not been mimicked in the laboratory. With unanesthetized animals, Abercrombie and Jacobs (1988) found that naloxone potentiated the increase in LC activity in response to stress but did not alter LC activity in a resting state. These results suggest that endogenous opioid inputs to the LC may not be tonically active during resting conditions but may become active during stress to exert a modulatory role on LC discharge activity. Further studies are required to determine whether opioid peptides are colocalized with excitatory transmitters such as gluta- mate and, if so, whether the afferent terminals differen- tially release these transmitters.

Some FOR-labeled dendrites received synaptic contacts from axon terminals lacking L-ENK immunoreactivity in the single sections examined, suggesting that opioid contain- ing afferents may be localized to portions of the plasma- lemma in a different section not included in our analysis. However, the unlabeled terminals, which sometimes con- tained large dense core vesicles, could contain another opioid peptide. A possible candidate is methionine (Met)- enkephalin which is also known to be an endogenous ligand of FOR. Further studies will be necessary to determine whether Met-ENK is found in a different population of axon terminals contacting FOR-labeled dendrites.

In our previous study, axon terminals forming synaptic contacts with pOR-labeled perikarya and dendrites con- tained numerous small clear vesicles and several large dense core vesicles and were often apposed to astrocytic processes. The location of the dense core vesicles along the perimeter of the plasma membranes is interesting in light of potential sites of transmitter release (Zhu et al., 1986). Our localization of FOR immunoreactivity to extrasynaptic portions of the plasmalemma of perikarya and dendrites suggests that the endogenous peptide may be released from

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sites distal to the zone of synaptic junction, as suggested for other brain regions (Zhu et al., 1986; Cheng et al., 1996). Glial processes may facilitate diffusion of the transmitter in the neuropil and thus affect postsynaptic modulation of the target (Pickel et al., 1995). Additional studies are necessary to test the hypothesis of extrasynaptic release of opioid peptides in the LC region.

ACKNOWLEDGMENTS The authors gratefully acknowledge the photographic

assistance of Ms. Joy Hornung and thank Dr. V.M. Pickel for comments on the manuscript. This work was supported by a Young Investigator Award from the National Associa- tion for Research on Schizophrenia and Depression, NIDA R29 DA09082, and an Established Investigator Award from the American Heart Association to E.J.V.B and NIH grant 18974, which was used to support some of the core equip- ment employed in the present study.

LITERATURE CITED Abercrombie, E.D., and B.L. Jacobs (1988) Systemic naloxone administra-

tion potentiates locus coeruleus noradrenergic neuronal activity under stressful but, not non-stressful conditions. Brain Res. 441:362-366.

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