α2A-adrenergic receptors in the rat nucleus locus coeruleus: subcellular localization in...

Ž .Brain Research 795 1998 157–169

Research report

a -adrenergic receptors in the rat nucleus locus coeruleus: subcellular2A

localization in catecholaminergic dendrites, astrocytes, and presynaptic axonterminals

Amy Lee a, Diane L. Rosin b, Elisabeth J. Van Bockstaele c,)

a Neuroscience Graduate Program, UniÕersity of Virginia, CharlottesÕille, VA 22908, USAb Department of Pharmacology, UniÕersity of Virginia, CharlottesÕille, VA 22908, USA

c Department of Pathology, Anatomy, and Cell Biology, Jefferson Medical College of Thomas Jefferson UniÕersity, Philadelphia, PA 19107, USA

Accepted 10 March 1998

Abstract

Ž . Ž .To define the anatomic substrates subserving the inhibitory actions of a -adrenergic receptors a -ARs in the locus coeruleus LC ,2 2Ž .we used dual-label immunoelectron microscopy with antibodies directed against the A-subtype of a -AR a -AR and the cate-2 2A

Ž . Ž .cholamine-synthesizing enzyme tyrosine hydroxylase TH . Of the profiles containing peroxidase labeling for a -AR a -AR-IR in2A 2AŽ . Ž . Ž . Ž .the LC ns735 , most were dendrites ;50% , glial processes ;30% , and axon terminals ;15% . a -AR-IR was also observed in2A

unmyelinated axons and perikarya. Within dendrites, a -AR-IR was associated with nonsynaptic regions of the plasma membrane and2A

subsurface cisternae. Approximately 60% of dendrites with a -AR-IR were dually labeled for TH. Fifty percent of the axon terminals2AŽ .contacting a -AR-immunoreactive dendrites formed asymmetric excitatory synaptic contacts. Axon terminals with a -AR-IR were2A 2A

not dually labeled for TH and generally formed asymmetric synapses with TH-immunoreactive dendrites that contained or lackeda -AR-IR. Astrocytic processes exhibiting a -AR-IR were closely apposed to TH-labeled dendrites. These results extend previous2A 2A

ultrastructural observations of a -ARs in the LC and suggest that the inhibitory actions of norepinephrine and epinephrine in this region2A

may be mediated by postsynaptic a -ARs on catecholaminergic dendrites and presynaptic a -ARs on excitatory inputs to2A 2A

catecholaminergic dendrites. In addition, the localization of a -AR-IR in astrocytic processes apposed to TH-immunoreactive dendrites2A

suggests a role for a -ARs in functional interactions between catecholaminergic dendrites and neighboring astrocytes. q 1998 Elsevier2A

Science B.V. All rights reserved.

Keywords: a -Adrenergic receptor; Locus coeruleus; Clonidine2A

1. Introduction

Many of the physiological effects of agonists at centralŽ .a -adrenergic receptors a -ARs are attributed to a -ARs2 2 2

in the major noradrenergic cell group, the locus coeruleusŽ .LC . Microinfusion of the a -AR agonist clonidine into2

w xthe LC of rats produces sedation 13 and reverses signs ofw xstress-induced behavioral depression 52 . In addition,

clonidine suppresses the activation of catecholaminemetabolism in the LC in response to hypotensive stimuliw x46 . The ability of clonidine to attenuate the behavioral

) Corresponding author. Department of Pathology, Anatomy, and CellBiology 1020 Locust Street, Rm. 526 Thomas Jefferson University,Philadelphia, PA 19107, USA. Fax: q1-215-923-3808; E-mail:[email protected]

w x w xchanges 57 and hyperactivation of LC neurons 1 follow-ing withdrawal from morphine has led to the use of a -AR2

agonists in the treatment of the physiological and psycho-logical symptoms caused by opiate withdrawal in humansw x24 .

Due to the clinical relevance of a -AR-mediated re-2

sponses in the LC, considerable research has focused onthe mechanisms underlying a -AR function in this region.2

Electrophysiologic studies indicate that clonidine adminis-tered systemically and iontophoretically inhibits the dis-

w xcharge rate of LC neurons 10,35 , an effect that is mim-icked by the endogenous a -AR ligands norepinephrine2

w xand epinephrine 9,54 . Subsequent intracellular recordingsfrom in vitro slice preparations showed that a -ARs hy-2

perpolarize LC neurons through activation of a Kq con-w xductance 62 . While high levels of a -AR binding sites2

0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.Ž .PII S0006-8993 98 00266-2

( )A. Lee et al.rBrain Research 795 1998 157–169158

w xand mRNA have been detected in the LC 37,41,51,63,64 ,receptor binding autoradiography and in situ hybridizationdo not provide the resolution required to characterize thesubcellular localization of a -ARs in this region. Consid-2

ering the widespread distribution of a -ARs in the brain2

and functional heterogeneity of inputs to the LC, identify-ing the subcellular sites of a -AR action is crucial for2

understanding how a -ARs modulate LC neuronal physiol-2

( )A. Lee et al.rBrain Research 795 1998 157–169 159

ogy. For example, since the LC receives a major excitatoryand inhibitory drive from cells that may also express

w xa -ARs in the rostral ventrolateral medulla 5,15,23,49 ,2

the inhibition of LC neuronal firing by clonidine may bemediated by a -ARs localized postsynaptically on LC2

neurons or presynaptically on excitatory afferents to theLC.

For resolving such issues, the immunocytochemical lo-calization of a -ARs offers distinct advantages over other2

receptor localization techniques. Using antibodies thatspecifically recognize particular subtypes of a -ARs in2

immunohistochemical preparations, our group has shownŽ .that immunoreactivity -IR corresponding to the A- andŽ .C-subtypes of a -AR a - and a -AR is associated2 2A 2C

w xwith most LC neurons 47,56 . Dual labeling with antibod-ies directed against the catecholamine synthesizing enzyme

Ž .tyrosine hydroxylase TH indicated that most of the cellsexhibiting a - and a -AR-IR were catecholaminergic2A 2Cw x32,49 . The localization of a -AR-IR in LC neurons is2A

of particular interest since many of the physiologic effectsof a -AR activation including sedation are attributed to2

w xthe A-subtype 33 . In addition, a -autoreceptors inhibit2A

norepinephrine release in major targets of LC efferentsw xsuch as the hippocampus and cerebral cortex 28,58 .

In a previous ultrastructural characterization of a -2Aw xAR-IR in the LC, Aoki et al. 4 reported that a -AR-IR2A

was associated almost exclusively with presynaptic termi-nals that were not catecholaminergic. However, the exis-tence of somatodendritic a -ARs is suggested by electro-2

w xphysiological studies 26 . The present study was under-taken to further detail the potential sites mediating a -AR2A

actions in the LC using dual immunocytochemical labelingfor a -ARs and TH in conjunction with electron mi-2A

croscopy. We demonstrate that a -AR-IR in the LC is2A

associated primarily with catecholaminergic dendrites,noncatecholaminergic presynaptic axon terminals, and as-trocytic processes. These results suggest that both pre- andpostsynaptic a -ARs may mediate the inhibitory effects2A

of catecholamines in the LC.

2. Materials and methods

2.1. Tissue preparation

ŽFive adult male Sprague–Dawley rats 200–250 g;.Hilltop Labs, Scottsdale, PA were used in this study. Rats

Žwere anesthetized with sodium pentobarbital 100 mgrkg,.i.p. and perfused transcardially through the ascending

aorta with 60 ml of 3.75% acroleinr2% paraformaldehydefollowed by 300 ml of 2% paraformaldehyder0.1 M phos-

Ž .phate buffer PB, pH 7.4 . Brains were removed and a 2mm coronal slice containing the LC was postfixed for 30min in 2% paraformaldehyderPB. Using a Vibratome,

Ž .sections 30 mm were cut through the rostrocaudal extentof the LC, collected into PB, and processed immediatelyfor immunocytochemistry. These procedures have beenapproved by the Thomas Jefferson University animal carefacility and conform to NIH guidelines.

2.2. Antisera

The characterization and specificity of the rabbit poly-clonal anti-a -AR antisera were described previously2Aw x49,56 . The antibodies were raised against a recombinant

Ž .fusion protein containing glutathione S-transferase GSTand 47 amino acids of the third intracellular loop of the rata -AR. The specificity of affinity-purified a -AR anti-2A 2A

bodies was demonstrated in western blot, immunoprecip-itation, and immunocytochemical analyses of cultured cellstransfected with the cDNA encoding the rat a -AR2Aw x49,56 . In addition, the a -AR immunoreaction product2A

in sections of rat brain was eliminated by preadsorption ofthe antibodies with the immunogenic fusion protein. TheTH antibody was a commercially available mouse mono-

Žclonal reagent directed against TH Incstar, Stillwater,.MN .

2.3. Immunocytochemistry

Vibratome sections were incubated in 1% sodium boro-hydriderPB, rinsed extensively in PB followed by 0.1 M

Ž .Tris-buffered saline TS, pH 7.4 , and incubated for 30Ž .min in 0.5% bovine serum albumin BSA rTS to block

non-specific antibody binding sites. Tissue sections werethen incubated for 36–48 h at 48C in rabbit polyclonal

Žanti-a -AR antibodies 10 mgrml, diluted in 0.1%2A.BSArTS . To remove anti-GST antibodies, a -AR anti-2A

bodies were preadsorbed to GST immobilized on sepharosebeads for 4 h at room temperature prior to use; beadsconjugated with the immunogenic a -AR fusion protein2A

Fig. 1. Electron micrographs showing the subcellular localization of a -AR-IR in perikarya, dendrites, and axon terminals in the LC. A: Peroxidase2AŽ . Ž . Ž . Ž .labeling for a -AR is associated with the plasma membrane short arrow , Golgi apparatus g short arrow , and subsurface cisternae long arrow of a2A

Ž . Ž . Ž .soma S with a visible nucleus n . The soma is apposed by a glial process asterisks . B: a -AR-IR is discretely localized near and on the plasma2AŽ . Ž . Ž . Ž . Ž .membrane short arrow of a dendrite D that is enveloped by labeled open straight arrow and unlabeled asterisks glial processes. C: A dendrite D

Ž . Ž .containing a -AR-IR receives an asymmetric synaptic contact open curved arrow from an unlabeled axon terminal ut . Peroxidase labeling for a -AR2A 2AŽ . Ž .is associated with a region of the plasma membrane short arrows apposed to a glial process asterisks . a -AR-IR can also be seen in an unmyelinated2A

Ž . Ž . Ž . Ž .axon ua . D: An axon terminal containing a -AR-IR A-t forms an asymmetric contact open curved arrow with an unlabeled dendrite D , both of2AŽ . Ž .which are partially enveloped by glial processes asterisks . The dendrite receives a second asymmetric contact open curved arrow from an unlabeled

Ž .axon terminal ut . ma, Myelinated axon. Scale bars0.5 mm.


Fig. 2. Localization of a -AR-IR in TH-labeled dendrites and their relationships with axon terminals and glial processes. A: Peroxidase labeling for2AŽ . Ž .a -AR short arrows is localized near the plasma membrane of a dendrite containing gold–silver labeling for TH TH-D that is enveloped by glial2AŽ . Ž . Ž .processes asterisks . D, unlabeled dendrite. B: a -AR-IR short arrows is associated with portions of the Golgi apparatus g , plasma membrane, and2A

Ž . Ž .vesicle-like structures near the plasma membrane of a TH-labeled dendrite TH-D apposed by three unlabeled axon terminals ut , one of which forms anŽ . Ž .asymmetric synaptic contact open curved arrow . Glial processes asterisks appose the dendrite and axon terminals. C: Peroxidase labeling for a -AR is2A

Ž . Ž . Ž .distributed within the cytoplasm long arrow of a TH-labeled dendrite TH-D receiving an asymmetric synaptic contact open curved arrow from anŽ . Ž . Ž .unlabeled axon terminal ut . An axon terminal containing a -AR-IR A-t, short arrows forms an asymmetric synaptic contact open curved arrow with2A

Ž . Ž .a TH-labeled dendrite TH-D . The labeled axon terminal and both dendrites are apposed by unlabeled glial processes asterisks . Scale barss0.4 mm inA, 0.5 mm in B, 0.45 mm in C.


were used in control experiments to determine levels ofnon-specific immunoreactivity in brain tissue. For duallabeling experiments, tissue sections were incubated with

Ž .anti-TH antibody 1:2000 in addition to a -AR antibod-2A

ies.The dual-labeling procedure was conducted according

w xto methods described by Chan et al. 11 . Sections incu-bated in the primary antibodies were rinsed in TS and

Žincubated with biotinylated goat anti-rabbit IgG 1:400,.Vector Laboratories, Burlingame, CA for 1 h at room

temperature. Sections were subsequently rinsed and incu-bated for 30 min in avidin–biotin horseradish peroxidase

Ž .complex Vector Laboratories . Peroxidase reaction prod-uct representing a -AR-IR was achieved by processing2A

sections with a 0.02% diaminobenzidine solution. Forgold–silver labeling of TH, sections were rinsed in 0.01 M

Ž .phosphate buffered saline PBS and incubated in a washbuffer containing 0.1% gelatinr0.8% BSArPBS for 10min. Sections were then incubated with a goat anti-mouse

ŽIgG conjugated to 1 nm gold particles 1:50 diluted in.wash buffer, Amersham, Arlington Heights, IL for 2 h at

room temperature, rinsed with wash buffer and subse-

quently with PBS. Following a 10 min incubation in 2%glutaraldehyderPBS, sections were rinsed in PBS and then

Ž .in 0.2 M sodium citrate buffer pH 7.4 . To visualizegold–silver labeling for TH, sections were exposed to

Ž .silver intensifying reagents Amersham for 3 to 6 min. Analternate set of tissue sections was processed for theperoxidase localization of TH and immunogold-silver iden-tification of a -AR-IR. To this end, the concentration of2A

the antiserum directed against a -AR was increased two2A

fold and a goat-anti-rabbit coupled to 1 nm gold particlesŽ .Amersham was employed as the secondary antiserum

Ž .followed by a silver enhancement procedure Amersham .For light microscopic evaluation of immunoreactivity,

tissue sections were mounted on gelatin-coated microscopeŽ .slides and coverslipped in DPX Aldrich, Milwaukee, WI .

For electron microscopy, sections were incubated in 2%osmium tetroxiderPB for 1 h, rinsed in PB, dehydrated,

Žand flat-embedded in Epon 812 Electron Microscopy.Sciences, Fort Washington, PA . An ultramicrotome was

Ž .used to obtain thin sections approximately 60–75 nm thatwere collected onto grids and counterstained with 5%uranyl acetate and subsequently Reynolds lead citrate.

Fig. 3. Immunogold silver labeling for a -AR-IR at extrasynaptic sites of a TH-labeled dendrite. A dendrite containing peroxidase labeling for TH2AŽ . Žcontains gold–silver deposits arrowheads for a -AR-IR on portions of the plasma membrane distal to the asymmetric synaptic specialization open2A

. Ž . Ž .curved arrow formed by an unlabeled terminal ut . A gold–silver particle can also be seen adjacent to a tubulovesicle long straight arrow . TheŽ .a -AR-IRqTH labeled dendrite is separated from a dendrite containing only peroxidase labeling for TH by an astrocytic process asterisks . Several2A

Ž .unlabeled dendrites uD can be identified in the neuropil. Bars0.5 mm.


2.4. Data analysis

Tissue sections corresponding to Plate 50 of the ratw xbrain atlas of Swanson 55 were processed for electron

microscopy. Thin sections were obtained from the regioncontaining the LC immediately adjacent to the fourthventricle. Due to the differential penetration of the peroxi-

w xdase and gold-labeled immunoreagents 11 , analysis wasrestricted to thin sections collected from the surface of thetissue and photomicrographs were taken only when bothperoxidase and gold–silver labeling were evident. Approx-imately 10 grids containing 5 to 10 thin sections wereanalyzed from at least three flat-embedded tissue sectionsfrom each rat. Immunoreactive profiles were classified

w xaccording to criteria described by Peters et al. 45 .Perikarya were defined by the presence of a nucleus, Golgiapparatus, and endoplasmic reticulum. Profiles were con-sidered dendrites if they contained uniformly distributedmicrotubules. Dendrites were defined as proximal if theycontained endoplasmic reticulum and were )0.7 mm indiameter. Axon terminals were distinguished by the pres-ence of synaptic vesicles and diameter greater than 0.1mm. Contacts between dendrites and axon terminals wereconsidered synaptic if a junctional complex was evident.

Ž .Synapses were classified as asymmetric Type I if a thickŽ .postsynaptic density was visible and symmetric Type II

w xif thin pre- and postsynaptic densities were observed 20 .Appositions were defined as closely spaced parallel plasmamembranes between profiles not separated by glial pro-cesses.

3. Results

At the ultrastructural level, a -AR-IR was observed2A

primarily in dendrites, axon terminals, and glial processesŽ .Figs. 1–5 using either peroxidase or gold–silver labelingtechniques. Similar results were obtained whether tissuesections were processed for single labeling with a -AR2A

antibodies or dual labeling with a -AR and TH-antibod-2A

ies. No peroxidase labeling resembling a -AR-IR was2A

observed by light or electron microscopy following pread-sorption of the antibodies with the a -AR fusion protein.2A

Gold–silver labeling for TH was primarily observed indendrites, perikarya, and axon terminals. This pattern oflabeling was not observed when the anti-TH antibody was

omitted from the dual labeling procedure. Of the totalŽ .a -AR-immunoreactive profiles examined n s 735 ,2A

Ž .51% were dendrites ns375 , 27% were glial processesŽ . Ž .ns199 , and 13% were axon terminals ns92 . a -2A

ŽAR-IR was also observed in unmyelinated axons -10%,. Ž .ns49 and perikarya -5%, ns20 .

3.1. Localization of a -AR-IR in catecholaminergic and2 A

noncatecholaminergic dendrites

Within perikarya and dendrites, a -AR-IR was local-2A

ized to portions of the Golgi apparatus and discrete regionsŽof nonsynaptic plasma membrane Fig. 1A–C, Fig. 2A,B,

.Figs. 3 and 4B . In addition, a -AR-IR was often associ-2A

ated with membranous structures in the cytoplasm includ-Ž .ing subsurface cisternae Fig. 1A and pleiomorphic

tubulovesicles characterized by electron lucent centersŽ .Figs. 3 and 5B . Dual-labeling experiments revealed thata -AR-IR and TH-IR were colocalized in a large propor-2A

Žtion of labeled dendrites Fig. 2A–C, Figs. 3 and 4B, Fig.. Ž .5B . Of the dendrites containing a -AR-IR ns206 in2A

Ž .dual-labeling experiments, 55% ns114 also exhibitedgold–silver labeling for TH.

Only a fraction of the dendrites containing a -AR-IR2AŽ .ns375 with or without TH-IR were apposed by axon

Ž .terminals 30%, ns115 . Of these dendrites, about 50%Ž . Žns58 received asymmetric synaptic contacts Fig. 1C,

.Fig. 2B,C, Figs. 3 and 4B, Fig. 5B . These axon terminalswere not labeled for TH and were characterized by smallclear synaptic vesicles and occasionally 1 or more dense

Ž .core vesicles Figs. 3 and 4B . Some of the axon terminalsforming asymmetric contacts with a -AR-labeled den-2A

Ždrites also contained peroxidase labeling for a -AR Fig.2A.5B . a -AR-labeled dendrites with or without TH-IR2A

generally did not receive symmetric contacts from presy-naptic axon terminals. The remaining axon terminals ap-posed to a -AR-labeled dendrites did not form a visible2A

synaptic contact in the plane of section that was examined.Most dendrites containing a -AR-IR were apposed by2A

Ž . Žglial processes 60%, ns222 that were unlabeled 76%,. Ž .ns168 or labeled for a -AR 24%, ns54 . These glial2A

processes often completely enveloped a -AR-labeled2AŽdendrites that were not apposed to axon terminals Fig. 1B,

.Fig. 2A . Glial processes were also apposed to dendritescontaining a -AR-IR and the axon terminals forming2A

ŽFig. 4. Localization of a -AR-IR in astrocytic processes apposed to TH-labeled dendrites. A: Numerous glial processes containing a -AR-IR open2A 2A. Ž .solid arrows appose dendrites exhibiting gold–silver labeling for TH TH-D . Solid curved arrow indicates a tight junction formed between glial

Ž . Ž .processes. ma, Myelinated axon. B: A glial process exhibiting filament bundles f and peroxidase labeling for a -AR open straight arrow apposes a2AŽ . Ž . Ž .TH-labeled dendrite TH-D also containing a -AR-IR short arrow . The dendrite receives an asymmetric synaptic contact open curved arrow from an2A

Ž . Ž .unlabeled axon terminal ut containing several dense core vesicles dcv . The axon terminal is also apposed by a glial process containing a -AR-IR2AŽ . Ž .open straight arrow . Peroxidase labeling for a -AR in a second TH-labeled dendrite TH-D is associated with the cytoplasm and plasma membrane2AŽ . Ž .short arrows . This dendrite is apposed by unlabeled glial processes asterisks . Scale barss0.7 mm in A, 0.4 mm in B.


ŽFig. 5. Associations between axon terminals containing a -AR-IR and TH-labeled dendrites. A: An axon terminal containing a -AR-IR A-t, short2A 2A. Ž . Ž . Ž .arrows and a dense core vesicle dcv forms a symmetric type synapse straight black arrow with a small unlabeled dendrite uD and is located near two

Ž . Ž . Ž .TH-labeled dendrites TH-D that are apposed by glial processes asterisks that exhibit peroxidase labeling for a -AR open straight arrows . One of the2AŽ . Ž .dendrites receives an asymmetric synaptic contact open curved arrow from an unlabeled axon terminal ut . a -AR-IR can also be seen in an2A

Ž . Ž . Ž .unmyleinated axon ua . B: Peroxidase labeling for a -AR short arrows is associated with an axon terminal A-t forming an asymmetric synaptic2AŽ . Ž .contact open curved arrow with a TH-labeled dendrite TH-D . Within the dendrite, peroxidase labeling for a -AR is associated with a large2A

Ž .tubulovesicle long arrow . Scale barss0.5 mm in A, 0.4 mm in B.

Žsynaptic contacts with these dendrites Fig. 2B,C, Figs. 3.and 4B .

3.2. Axon terminals exhibiting a -AR-IR form asymmet-2 A

ric synapses with TH-immunoreactiÕe dendrites

a -AR-IR was localized in axon terminals that usually2AŽformed asymmetric contacts with dendrites ;75%, ns50

. Ž .of 66 labeled terminals Fig. 1D, Fig. 2C, Fig. 5B . Lessthan 1% of the a -AR-IR axon terminals formed sym-2A

Ž .metric type synapses Fig. 5A . The a -AR-labeled axon2A

terminals were not dually labeled for TH, but the post-synaptic targets of these axon terminals usually contained

Ž . Ž .TH-IR 90%, ns45 Fig. 2C, Fig. 5B . Both the presy-naptic a -AR-labeled terminal and the postsynaptic den-2A

Ždrite were often apposed by glial processes Fig. 1D, Fig..2C . Within axon terminals, a -AR-IR was either dis-2A

Žtributed diffusely around synaptic vesicles Fig. 1D, Fig..5A,B or restricted to regions of the plasma membrane that

Žwere away from or close to a synaptic junction Fig. 1D,.Fig. 2C, Fig. 5B . Most axon terminals containing a -2A

AR-IR had small clear synaptic vesicles and occasionallyŽ .dense-core vesicles Fig. 5A .

3.3. a -AR-IR is localized in astrocytic processes ap-2 A

posed to dendrites containing TH-IR

In addition to localization in dendrites and axon termi-nals, a -AR-IR was found in glial processes. These2A

processes exhibited irregular contours characteristic of as-Ž .trocytes Fig. 1B, Fig. 4A,B, Fig. 5A . Peroxidase labeling

for a -ARs was distributed on the plasma membrane of2AŽ .glial processes Fig. 4A, B, Fig. 5A but was also associ-

ated with the cytoplasm in cases where a larger portion ofŽ .the glial profile was visible Fig. 1B, Fig. 4A,B . In some

glial profiles exhibiting bundles of filaments, a -AR-IR2A

was localized in portions of rough endoplasmic reticulumŽ .not shown . In dual-labeling experiments, a large percent-age of the glial processes exhibiting a -AR-IR were2A

Žapposed to TH-immunoreactive dendrites ;50%, ns73. Ž .of 150 labeled glial processes Fig. 4A,B, Fig. 5A . These

glial processes often completely enveloped TH-labeledŽ .dendrites Fig. 4A .


4. Discussion

The present study demonstrates that a -AR-IR is pri-2A

marily localized in TH-labeled dendrites, as well as axonterminals and astrocytes in the LC. Although the existenceof presynaptic a -AR-IR is consistent with results de-2A

w xscribed in a previous report 4 , our study provides the firstultrastructural evidence for prominent localization of a -2A

AR-IR in catecholaminergic dendrites as well as in astro-cytes in the LC. Thus, a -ARs may serve both pre- and2A

postsynaptic functions in this region. In addition, the local-ization of a -AR-IR in astrocytic processes apposed to2A

TH-immunoreactive dendrites suggests a role for gliala -ARs in functional interactions between catecholamin-2A

ergic dendrites and neighboring astrocytes.

4.1. Methodological considerations

Immunoreaction product resulting from processing brainsections with the a -AR antibodies is referred to as2A

‘a -AR-immunoreactivity’ since the extent to which the2A

antibodies recognize proteins other than a -ARs is not2A

known. However, the immunocytochemical labeling de-scribed in our study is likely to represent the subcellulardistribution of a -ARs for a number of reasons. First, our2A

group has extensively characterized the specificity of thea -AR antibodies used in the present study by immuno-2A

blotting, immunoprecipitation, and immunocytochemicalw xanalyses 49,56 . The antibodies recognized a protein of

the predicted molecular weight of the a -AR from cells2A

transfected with the DNA encoding the a -AR but not in2A

untransfected cells or cells transfected with the DNAencoding the B- or C-subtypes of a -AR. Second, our2

immunohistochemical maps of a -AR-IR in the rat brain2A

correspond with results obtained by other groups usingreceptor binding autoradiography and in situ hybridizationw x37,41,51,63 . In particular, the reportedly high levels ofa -AR mRNA in the LC correlate with the prominent2A

w xlocalization of a -AR-IR in this region 49,56 . Third,2Aw xconsistent with our previous studies 49,56 , we did not

observe a -AR-IR in the LC following preadsorption of2A

the antibodies with the a -AR immunogen at the light or2A

electron microscopic levels, further underscoring the speci-ficity of the antibodies for the immunogen.

Due to reports that the immunogold-silver method isw xless sensitive than peroxidase-based methods 11 , struc-

tures containing relatively low levels of TH may not havebeen included in our analysis. At the same time, thecomparatively low-intensity of a -AR-IR with respect to2A

TH-IR suggests that even with the enhanced sensitivityprovided by the avidinrbiotin peroxidase labeling method,structures containing low levels of a -ARs may not have2A

been detected. However, the possibility that quantitation ofdually labeled profiles was biased by differential sensitivi-ties of the labeling methods was reduced by restricting theanalysis to the most superficial thin sections where im-

munolabeling with both markers was most intense anddetectable in numerous profiles.

4.2. Subcellular localization of a -AR-IR in perikarya2 A

and dendrites

The association of a -AR-IR with the Golgi apparatus2A

and subsurface cisternae within LC perikarya and dendritesmay represent various stages in the biosynthetic processingof the receptor. a -ARs, like most G-protein coupled2A

w xreceptors, are glycoproteins 30 which could explain theassociation of a -AR-IR with organelles such as the2A

Golgi apparatus where glycosylation of membrane proteinsw xtakes place 7 .

In previous light microscopic studies, we and othershave demonstrated that a -AR-IR in the LC was gener-2A

w xally punctate and restricted to cell somata 4,49,56 . Ultra-structural studies indicate that this pattern of a -AR-IR2A

may correspond to large clusters of peroxidase labelingassociated with multivesicular bodies or portions of the

w xendoplasmic reticulum and Golgi apparatus 4,31,48 . Inthe present study, these large clusters of a -AR-IR were2A

generally not typical of perikaryal labeling at the light orelectron microscopic levels. However, this pattern of im-munolabeling was more prevalent in preliminary experi-ments where low levels of detergent were used, suggestingthat detection of the intracellular clustering of a -AR-IR2A

in perikarya is enhanced by membrane permeabilization.

4.3. Localization of a -AR-IR in TH-labeled dendrites2 A

The prominent distribution of a -AR-IR in TH-labeled2A

dendrites is consistent with observations that somatoden-dritic a -ARs mediate inhibitory effects on LC neuronal2

w xexcitability 12,14,26,27,44 . The presence of a -AR-IR2A

in dendrites lacking TH immunoreactivity suggests thatcatecholaminergic modulation of this region may also in-

w xvolve non-LC dendrites which extend into the LC 53 .Activation of dendritic a -ARs may hyperpolarize LC2A

neurons and inhibit their spontaneous discharge throughq w xthe activation of K channels 62 . In addition, LC den-

drites may be the site where a -ARs and m-opioid2A

receptors couple to the same Kq conductance via pertussisw xtoxin sensitive G-proteins 2,3,43 . This possibility is

strengthened by our observations that adrenergic and opi-oid containing axon terminals converge on common den-

w xdrites in the LC 59 and that a -AR-IR is localized in2A

TH-immunoreactive dendrites receiving asymmetric con-tacts from unlabeled axon terminals, a distribution thatparallels that of m-opiate receptors in a previous ultrastruc-

w xtural study 61 . Since asymmetric synapses are correlatedw xwith excitatory neurotransmission 8,45 , both a -ARs2A

and m-opiate receptors localized postsynaptically maymodulate the activation of LC neurons by excitatory affer-ents, a mechanism that may account for the efficacy of


a -AR agonists such as clonidine in the treatment of opiate2w xwithdrawal symptoms 1,24,57 .

Although none of the dendrites containing a -AR-IR2A

received synaptic contacts from axon terminals containingTH-IR, it should be noted that, due to the limited sensitiv-ity of the gold–silver labeling method for TH, the actualdistribution of TH-immunoreactive axon terminals mayhave been underestimated. Thus, it is possible that some ofthe unlabeled terminals that formed synaptic contacts witha -AR-immunoreactive dendrites may actually have been2A

catecholaminergic. However, several lines of evidencesuggest that dendritic a -ARs may be activated by cate-2A

cholamines released nonsynaptically in the LC. First, mostdually labeled dendrites were apposed to astrocytes orother TH-immunoreactive dendrites. In fact, only a minor-

Ž .ity 30% of dendrites containing a -AR-IR were ap-2A

posed to axon terminals which suggests that most dendritica -ARs may not be activated by catecholamines released2A

at synaptic junctions. Second, in dendrites that did receivesynaptic contacts, we observed peroxidase labeling fora -ARs on non-junctional regions of the plasma mem-2A

brane. The nonsynaptic localization of a -AR-IR in TH-2A

labeled dendrites suggests that dendritic a -ARs may be2A

activated by catecholamines that diffuse extracellularlyw xfrom distant sites of release via volume transmission 19 .

Our findings that nearly half of all a -AR-labeled2A

profiles in the LC are either catecholaminergic or noncate-cholaminergic dendrites differ from those of Aoki et al.w x4 , who found a -AR-IR in axon terminals but few2A

dendrites in the LC. The discrepancy may be due todifferences in the a -AR antibodies used in the two2A

studies. Although both reagents are rabbit polyclonal anti-bodies directed against overlapping regions of the intra-

w xcellular third loop of the a -AR 29,49 , they are not2A

identical as indicated by some differences in immunolabel-ing patterns produced by the antibodies in various regions

w xof the brain 4,56 . Thus, dendritic a -ARs may be2A

processed or inserted into the plasma membrane such thatepitopes recognized by our a -AR antibodies are readily2A

accessible. Alternatively, methodological differences, suchas the use of low levels of detergent or freeze thawingprocedures, may have altered the antigenicity of dendritica -ARs such that they were not detected in the previous2A

w xstudy 4 .

4.4. a -AR-IR in axon terminals forming asymmetric2 A

contacts with TH-labeled dendrites: potential sites ofpresynaptic modulation of LC neurons

Despite suggestions that presynaptic a -ARs on recur-2

rent collaterals could mediate the autoinhibition of nor-w xepinephrine release in the LC 26,27 , we did not find

evidence for the anatomic correlate of presynaptic a -au-2

toreceptors in our study. While axon terminals comprised afraction of the a -AR-immunoreactive profiles in the LC,2A

none of the axon terminals containing a -AR-IR were2A

dually labeled for TH. Due to the limited sensitivity of thegold–silver labeling method for TH, we cannot exclude thepossibility that some dually labeled axon terminals wereunderrepresented in our analysis. Interestingly, Aoki et al.w x4 also found that most of the axons and axon terminalscontaining a -AR-IR lacked immunogold labeling for the2A

catecholamine synthesizing enzyme, dopamine b-hydrox-ylase. In the hippocampus, a brain region whose solesource of noradrenergic innervation derives from the LC,

w xMilner et al. 39 reported a low incidence of co-localiza-tion between a -AR and TH in axon terminals. These2A

results are also surprising in light of results from electro-physiological studies which predicted presynaptic modula-tion of norepinephrine release in this brain region. Thus,the association of a -AR-IR with non-catecholaminergic2A

axon terminals may be representative of the distribution ofpresynaptic a -ARs in the LC.2A

The a -AR-IR in axon terminals forming predomi-2A

nantly asymmetric synapses may derive from the rostralw xventrolateral medulla 15,16 . Electrical stimulation of this

region significantly enhances the discharge rate of LCneurons through the release of excitatory amino acids

w xwithin the LC 17 . In recordings from brain slices in vitro,the stimulation of LC neurons by glutamate is significantlyenhanced in the presence of a -AR antagonists, which2

suggests that norepinephrine released from collateralswithin the LC in response to excitatory stimuli inhibits LC

w xneurons via activation of a -ARs 26 . Although the colo-2

calization of a -AR-IR and TH-IR in dendrites suggests2A

that postsynaptic a -ARs may mediate the autoinhibition,2A

the asymmetric synapses formed by a -AR-labeled axon2A

terminals suggests that the a-subtype may modulate excita-tory inputs to LC neurons from a presynaptic locus as well.This possibility is supported by evidence obtained from insitu hybridization and immunohistochemical studies indi-cating that neurons in the rostral ventrolateral medulla

w xexpress a -ARs 49,63 .2A

The localization of a -AR-IR in axon terminals of2A

excitatory afferents may also explain functional distinc-tions between a -ARs and opiate receptors in the LC.2

Although both opiate receptors and a -ARs activate the2w xsame effector systems in LC neurons 2,3,43 , Simson et

w xal. 52 demonstrated that antagonism of a -ARs but not2

opiate receptors augments the responsiveness of LC neu-rons to excitatory stimulation. Noradrenergic activation ofpresynaptic a -ARs may regulate the release of excita-2A

tory neurotransmitters in the LC such that blockade ofthese presynaptic a -ARs would enhance the efficacy of2A

excitatory stimuli.

4.5. Astrocytic a -AR-IR and relation to TH-labeled den-2 A

drites

The localization of a -AR-IR in glial processes ap-2A

posed to TH-immunoreactive dendrites may support a rolefor astrocytes as targets for circulating catecholamines in


the LC. The functional significance of astrocytic a -ARs2

is suggested by reports that a -ARs regulate intracellular2w x w xcalcium 50 and cAMP levels in glial cell cultures 36 .

These signaling pathways may mediate changes in astro-w xcytic morphology 40 that, by promoting the insertion or

withdrawal of astrocytic processes between dendrites andaxons, could influence interneuronal communication withinthe LC. The ensheathment of a majority of LC dendritesby astrocytes, demonstrated in the present and previous

w xultrastructural studies 22,59,61 , underscores the potentialimportance of astrocytes in this region. In addition, thesimilar distribution of NMDA receptors in astrocytic pro-

w xcesses near TH-labeled dendrites in the LC 60 suggeststhat glial functions may be coordinately regulated by nor-epinephrine and glutamate.

4.6. Functional implications

The importance of the LC in the control of vigilance,attention, and adaptive responses to stress is well-docu-

w xmented 6 . Many of these functions are influenced by theactivation of a -ARs in the LC. The sleep-inducing effect2

of clonidine infusions into the LC of rats suggests that thesedation that often accompanies the use of clonidine as anantihypertensive agent is mediated by a -ARs in the LC2w x13,21 . The beneficial effects of clonidine in the treatmentof attention-deficit hyperactivity disorder in children maybe due to the inhibitory actions of this drug on LC outputw x25 . Clonidine blocks the stress-induced hyperactivation

w xof LC neurons 46 , and a -AR-binding sites are signifi-2

cantly downregulated in the LC following chronic psy-w xchosocial stress 18 . Our results indicating the widespread

distribution of a -AR-IR in a variety of subcellular ele-2A

ments in the LC support previous evidence obtained frompharmacologic and gene-targeting studies suggesting thata -ARs mediate many of the physiologic effects of cloni-2A

w xdine 34,38 . In addition, the localization of a -AR-IR in2A

non-catecholaminergic axon terminals in addition to cate-cholaminergic dendrites suggests that the activity of LCneurons, and subsequent output of the noradrenergic sys-tem, may be dually regulated by pre-and postsynaptica -ARs in the LC.2A

Acknowledgements

The authors gratefully acknowledge Amy Wissekerkeand Eric Colago for expert technical assistance, SuzanneBotkin for helpful technical advice, and James Peoples forphotographic assistance. Supported by grants from NIHŽ .F31 MH11074-01 to AL , the American Heart Associa-

Žtion 95011200 to Kevin R. Lynch and 96002240 to.EJVB , the Scottish Rite Schizophrenia Research Program

Ž . Žto DLR and NIDA R29 DA09082 and RO3 DA10450 to. Ž .EJVB , and NIMH 40008 to EJVB .

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