Distribution of ?1A adrenergic receptor m RNA in the rat brain visualized by in situ hybridization

Distribution of a1A Adrenergic ReceptormRNA in the Rat Brain Visualized by

In Situ Hybridization

ANNA V. DOMYANCIC AND DAVID A. MORILAK*Department of Pharmacology, University of Texas Health Science Center at San Antonio,

San Antonio, Texas 78284-7764

ABSTRACTNorepinephrine has been implicated in a number of physiological, behavioral, and

cellular modulatory processes in the brain, and many of these modulatory effects areattributable to a1 adrenergic receptors. At least three a1 receptor subtypes have beenidentified by molecular criteria, designated a1A, a1B, and a1D. The distributions of a1B and a1Dreceptor mRNA expression in rat brain have been described previously, but the cDNA for therat a1A receptor has only recently been cloned and characterized. In the present study, we useda radiolabelled riboprobe derived from the rat a1A receptor cDNA to describe the distributionof a1A message expression in the rat brain. The highest levels of a1A adrenergic receptor mRNAexpression were seen in the olfactory bulb, tenia tectae, horizontal diagonal band/magnocellular preoptic area, zona incerta, ventromedial hypothalamus, lateral mammillarynuclei, ventral dentate gyrus, piriform cortex, medial and cortical amygdala, magnocellularred nuclei, pontine nuclei, superior and lateral vestibular nuclei, brainstem reticular nuclei,and several cranial nerve motor nuclei. Dual in situ hybridization combining a radioactiveriboprobe for choline acetyltransferase mRNA with a digoxigenin-labeled a1A riboprobe in thefifth and seventh cranial nerve motor nuclei showed that the a1A mRNA is expressed incholinergic motor neurons. Prominent a1A hybridization signal was also seen in the neocortex,claustrum, lateral amygdala, ventral cochlear nucleus, raphe magnus, and in the ventral hornof thoracic spinal cord. This overall pattern of expression, considered in comparison with thatpreviously described for the other a1 adrenergic receptor subtypes, may shed light on thedifferent roles of the a1 receptors in mediating the neuromodulatory effects of norepinephrinein processes such as arousal, neuroendocrine control, sensorimotor regulation, and the stressresponse. J. Comp. Neurol. 386:358–378, 1997. r 1997 Wiley-Liss, Inc.

Indexing terms: a1 adrenoreceptors; neuroanatomy; norepinephrine; receptor subtypes; riboprobe

The monoaminergic neurotransmitter norepinephrine(NE) has been implicated in a variety of complex behav-ioral and physiological functions such as cardiovascularand neuroendocrine control, state-dependent modulationof motor activity, behavioral arousal, and the CNS re-sponse to stress. Many of the behavioral, physiological,and cellular modulatory effects of norepinephrine havebeen attributed to postsynaptic a1 adrenergic receptors(Aghajanian and Rogawski, 1983; Waterhouse et al., 1991).Pharmacological binding studies have shown the existenceof at least two high affinity a1 receptor binding sites in therat brain, designated a1A and a1B (Morrow and Creese,1986). The distribution of a1 receptors in brain have beendescribed in radioligand binding studies, typically employ-ing 3H-prazosin or 125I-HEAT as ligands (Jones et al., 1985;Young and Kuhar, 1980), which do not discriminate be-tween the different a1 receptor classes. Early studies using

agents that are capable of coarsely differentiating a1A-likefrom a1B binding in membrane preparations have sug-gested that the a1A-like binding sites predominate inhippocampus, while a1B binding is highest in thalamus,with both types present in the cerebral cortex (Wilson andMinneman, 1989). Such agents have limited utility fordescribing with any resolution the regional distribution ofa1 receptor subtypes by autoradiography because of very

Grant sponsor: American Heart Association; Grant number: 95G-375;Grant sponsor: The Whitehall Foundation; Grant number: S95-17; Grantsponsor: NIMH; Grant number: MH 53851.

*Correspondence to: David Morilak, Ph.D., Department of Pharmacology,University of Texas Health Science Center, 7703 Floyd Curl Drive, SanAntonio, TX 78284-7764. E-mail: [email protected]

Received 1 May 1996; Revised 11 April 1997; Accepted 30 April 1997

THE JOURNAL OF COMPARATIVE NEUROLOGY 386:358–378 (1997)

r 1997 WILEY-LISS, INC.

low signal strength or interactions with other transmitterreceptors (Laporte et al., 1991).

More recently, the cloning of cDNAs encoding a1 adrener-gic receptors has suggested the existence of at least threerecombinant subtypes, and considerable confusion hasexisted regarding the correspondence between the clonedcDNA sequences and endogenous receptor subtypes asdefined by pharmacological criteria. Much of the uncer-tainty in receptor classification has arisen from apparentspecies differences in receptor characteristics, and fromthe fact that the pharmacologic properties of recombinantreceptors expressed in transfected cell lines can differsubstantially from those of the corresponding native recep-tors in vivo (reviewed in Hieble et al., 1995). As theseuncertainties have been clarified, a consensus has emerged(Hieble et al., 1995). The cloned a1b receptor cDNA corre-sponds to the pharmacologic a1B subtype (Cottechia et al.,1988; Voigt et al., 1990), while pharmacologic a1A-like

binding appears to be comprised of two closely-relatedreceptor subtypes. A cDNA clone derived from bovinetissue was originally designated a1c because it was be-lieved to represent a novel subtype (Schwinn et al., 1990).However, the homologous sequence cloned from rat tissuehas clearly been shown to correspond to the classical a1Apharmacologic subtype, and has now been correctly identi-fied as such (Perez et al., 1994; Rokosh et al., 1994). A thirddistinct a1 receptor, originally misclassified as the a1Asubtype because it possesses very similar pharmacologicalcharacteristics (Lomasney et al., 1991; Perez et al., 1991;Schwinn and Lomasney, 1992), has now been designateda1D.

Because of their extreme pharmacological similaritiesand the lack of clearly discriminating ligands, it has notbeen possible to differentiate the endogenous a1A and a1Dreceptor subtypes by radioligand binding approaches. How-ever, the availability of cDNA sequence information and

Abbreviations

3 oculomotor nucleus4 trochlear nucleus7 facial nucleusac anterior commissureaca anterior commissure, anteriorAcb nucleus accumbensACo anterior cortical amygdaloid nucleusAHi amygdalohippocampal areaAHP anterior hypothalamic area, posteriorAOB accessory olfactory bulbAOM anterior olfactory nucleus, medialAOP anterior olfactory nucleus, posteriorAOV anterior olfactory nucleus, ventralArc arcuate nucleusBL basolateral amygdaloid nucleusBLP basolateral amygdaloid nucleus, posteriorBST bed nucleus of the stria terminalisCA1-3 fields CA1-3 of Ammon’s hornCe central amygdaloid nucleusCeL central amygdaloid nucleus, lateralCeM central amygdaloid nucleus, medialCg cingulate cortexCG central (periaqueductal) grayCGLV central gray, lateral ventralChAT choline acetyltransferaseCl claustrumCNS central nervous systemCo cortical amygdaloid nucleuscp cerebral peduncleCPu caudate putamenDEn dorsal endopiriform nucleusDEPC diethyl pyrocarbonateDG dentate gyrusDGv dentate gyrus, ventralDk nucleus DarkschewitschDLL dorsal nucleus of the lateral lemniscusDM dorsomedial hypothalamic nucleusDR dorsal raphe nucleusDTg dorsal tegmental nucleusEPl external plexiform layer of olfactory bulbf fornixfr fasciculus retroflexusFr frontal cortexg7 genu of the facial nerveGi gigantocellular reticular nucleusGiA gigantocellular reticular nucleus, alphaGl glomerular layer olfactory bulbHDB horizontal limb of the diagonal band nucleus125I-HEAT [125I]-(2-b(4-hydroxyphenyl)-ethyl-aminomethyl)-tetraloneIC inferior colliculusIGr internal granular layer of the olfactory bulbIP interpeduncular nucleusIPl internal plexiform layer olfactory bulbIRt intermediate reticular nucleus

KF Kolliker-Fuse nucleusLa lateral amygdaloid nucleusLat lateral cerebellar nucleusLH lateral hypothalamic areaLHb lateral habenulaLM lateral mammillary nucleusLO lateral orbital cortexLPB lateral parabrachial nucleusLPO lateral preoptic areaLSO lateral superior oliveLVe lateral vestibular nucleusMCPO magnocellular preoptic nucleusMe medial amygdaloid nucleusMe5 mesencephalic trigeminal nucleusMeA medial amygdaloid nucleus, anteriorMeP medial amygdaloid nucleus, posteriorMG medial geniculate nucleusMo5 motor trigeminal nucleusMP medial mammillary nucleus, posteriorMPB medial parabrachial nucleusNE norepinephrineOc occipital cortexox optic chiasmPar parietal cortexPBS phosphate-buffered salinePCRtA parvocellular reticular nucleus, alphaPir piriform cortexPLCo posterolateral cortical amygdaloid nucleusPMCo posteromedial cortical amygdaloid nucleusPn pontine nucleiPnC pontine reticular nucleus, caudalPnO pontine reticular nucleus, oralpy pyramidal tractRF reticular formationRMC red nucleus, magnocellularRMg raphe magnus nucleusRt reticular thalamic nucleusscp superior cerebellar peduncleSNC substantia nigra, compactSNR substantia nigra, reticularSp5 spinal trigeminal nucleusSSC sodium chloride/sodium citrate bufferSuVe superior vestibular nucleusT2-4 thoracic spinal cord, segments 2-4Te temporal cortexTT tenia tectaTz nucleus of the trapezoid bodyV third ventricleVCA ventral cochlear nucleus, anteriorVDB vertical limb of the diagonal band nucleusVMH ventromedial hypothalamic nucleusVP ventral pallidumZI zona incerta

a1A ADRENERGIC RECEPTOR mRNA IN RAT BRAIN 359

the cloned cDNAs themselves have made it possible todifferentiate and localize the distribution of the threeclosely related a1 adrenergic receptor subtypes by using insitu hybridization to visualize expression of their respec-tive mRNA. Recent studies using labeled oligonucleotideshave described the differential distribution of the a1B anda1D receptor subtypes in the brain (McCune et al., 1993;Pieribone et al., 1994), and the results of these studieshave been in good general agreement with previous radio-ligand binding studies. However, because the classificationof the a1 receptor subtypes has only recently been clarified,and the rat homolog of the a1A receptor cDNA has onlyrecently been cloned and characterized (Perez et al., 1994),the distribution of a1A receptor mRNA has not yet beendescribed in brain. Thus, in the present study, we exam-ined the distribution of a1A receptor message expression inthe rat brain by using a radiolabeled riboprobe generatedfrom the rat a1A receptor cDNA.

MATERIALS AND METHODS

Production of a1A receptor riboprobe

Plasmid pMT28-Ra1A, including the entire coding regionof the rat a1A receptor cDNA (Perez et al., 1994), wasobtained from Dr. Dianne Perez, Cleveland Clinic Founda-tion. A 604 bp Xho I-Not I fragment was subcloned intopBluescript II to generate plasmid pRa1A-XN, represent-ing nt 858-1461 in the C-terminal coding region and38-UTR of the a1A mRNA. The antisense riboprobe used forin situ hybridization was transcribed by using T7 RNApolymerase after linearizing the template cDNA plasmidwith the restriction enzyme Xho I, while the correspondingcontrol sense strand probe was transcribed with T3 poly-merase following linearization with Not I. Radiolabeledriboprobes were synthesized by using a commerciallyavailable transcription kit (Stratagene, La Jolla, CA)according to the manufacturers directions, with the addi-tion of a-[35S]-UTP (New England Nuclear/Dupont, Bos-ton, MA), without addition of any cold UTP, to a specificactivity of 2 x 109 cpm/µg.

In situ hybridization

All animal procedures were in accordance with the NIHGuide for the Care and Use of Laboratory Animals, and allprotocols have been approved by the Institutional AnimalCare and Use Committee of the University of Texas HealthScience Center at San Antonio.

Seven adult male Sprague-Dawley rats (Harlan, India-napolis, IN) were killed by rapid decapitation. The brainswere removed, frozen immediately by immersion in isopen-tane on dry ice, and stored less than 1 month at -70°C untiluse. Twenty micron brain sections were cut on a cryostat,and two sections out of every ten were thaw-mounted ontopoly-L-lysine coated microscope slides, two sections perslide. Adjacent control sections were collected in everyfourth series for hybridization with the sense-strand probe.

All solutions used for prehybridization and hybridiza-tion were treated with diethyl pyrocarbonate (DEPC) andautoclaved or filter sterilized. Slide-mounted sections werefixed in 4% paraformaldehyde for 15 minutes, rinsed inPBS, acetylated in 0.25% acetic anhydride/0.1 M triethanol-amine, pH 8.0, and rinsed in 2X SSC (1X SSC is 150 mMsodium chloride, 15 mM sodium citrate, pH 7.2). Sectionswere then dehydrated in an ascending series of ethanol

washes, delipidated in chloroform, and air-dried beforehybridization.

Hybridization buffer consisted of 50 mM sodium phos-phate, 3X SSC, 5X Denhardt’s solution, 0.1 mg/ml salmonsperm DNA, 0.1 mg/ml yeast tRNA, 10 mM dithiothreitol,10% dextran sulfate, and 50% deionized formamide. Sec-tions were hybridized, two per slide under glass coverslips,in 60 µl of hybridization buffer to which radiolabeled senseor antisense riboprobe was added to a final concentrationof 4 x 107 cpm/ml (approximately 20 ng/ml). They werethen placed on racks and incubated overnight in a sealedhumidified chamber at 60°C.

All posthybridization solutions contained 10 mM b-mer-captoethanol. After hybridization, excess probe was re-moved by rinsing in four washes of 2X SSC, then digestingthe sections with RNAse A (20 µg/ml, 30 minutes at 37°C).They were then taken through a series of rinses ofincreasing stringency: 10 minutes each in 1X, 0.5X, 0.2X,and 0.1X SSC, followed by 2 x 1 hour washes and anovernight wash in 0.1X SSC, 15% formamide at 60°C.Sections were then rinsed in 1X SSC, dehydrated andapposed to Kodak Biomax X-ray film for 3–5 days. Theslides were then dipped in Kodak NTB-2 emulsion, ex-posed at 4°C for 10–14 days, and developed by standarddarkroom chemistry using Kodak D19 developer. In somecases, the hybridized sections were then lightly counter-stained in Cresyl Violet and coverslipped for darkfieldmicroscopy and photomicrography. In other cases, adja-cent sections were stained for regional definition andmatched to the hybridized slides.

Dual in situ hybridization

In three additional animals, we determined whetherneurons expressing a1A receptor mRNA in the fifth andseventh cranial nerve motor nuclei were cholinergic motorneurons. This was done by double in situ hybridization,combining a non-radioactive digoxigenin-labeled a1A ribo-probe with a [35S]-labeled probe for choline acetyltransfer-ase (ChAT), the synthetic enzyme for acetylcholine (cDNAprovided by Dr. C. Gall, University of California, Irvine;Lauterborn et al., 1993). The ChAT probe was synthesizedas described above, and the non-radioactive a1A cRNAprobe was synthesized incorporating digoxigenin-11-UTP,according to the manufacturer’s instructions (Boehringer-Mannheim, Indianapolis, IN). Tissue preparation andhybridization were as described above, with the addition ofa 10 minute digest in proteinase K (1 µg/ml) at 37°C priorto acetylation. The two probes were applied together ontobrainstem sections through the fifth and seventh cranialnerve motor nuclei. The digoxigenin-labeled a1A probe wasdiluted to 7 µg/ml.

Posthybridization washes were as above. The digoxi-genin-labeled a1A riboprobe was visualized using a commer-cially available detection kit (Genius 3, Boehringer-Mannheim), according to manufacturer’s instructions, withminor modifications. After the final posthybridization SSCwash, sections were rinsed in PBS and pre-incubated inPBS with 3% normal sheep serum and 0.3% Triton X-100.They were then incubated overnight at 4°C in alkaline-phosphatase-conjugated rabbit anti-digoxigenin antibody,diluted 1/1,000 in the same blocking solution. The follow-ing day, sections were rinsed, pre-incubated in solution 3,and an alkaline-phosphatase reaction was initiated withnitro blue tetrazolium and X-phosphate as substrates. Thereaction was allowed to proceed in the dark for 5–7 hours,

360 A.V. DOMYANCIC AND D.A. MORILAK

with periodic monitoring, until a dark purple reactionproduct was seen on the antisense sections, with minimalbackground or nonspecific reaction product on the sensecontrol slides. The reaction was terminated by rinsing for 5minutes in solution 4. After rinsing, the reaction productwas fixed for 1 hour in 2.5% glutaraldehyde, followed byseveral rinses in 1X SSC. Sections were then dehydratedand apposed to Kodak Biomax X-ray film for 2 days for filmautoradiography. The slides were then dipped in IlfordK5.D nuclear track emulsion, exposed at 4°C for 7 days,and developed by standard darkroom chemistry usingKodak D19 developer. Sections were then coverslippedunder glycerol for light microscopy and photomicrography.Controls included incubation of the ChAT antisense probealong with the digoxigenin-a1A sense probe, and the a1Aantisense with the ChAT sense probe. Also, a1A-expressingcells in other regions of the same sections that were notexpected to show ChAT expression, including the lateralvestibular nucleus and the nucleus raphe magnus, wereexamined to rule out nonspecific accumulation of silvergrains on the alkaline-phosphatase reaction product.

Analyses

Film autoradiographs were analyzed using a 35 mmslide scanner adapted to accomodate microscope slides orautoradiographic films cut to 1 x 3 inches (Polaroid Sprint-Scan35 and PathScan Enabler, Meyer Instrument Co.,Houston, TX). Images of matched cresyl-stained sectionswere likewise scanned and traced to create the line-drawings in Figure 1, and as an aid to determiningregional localization of the a1A receptor message.

Emulsion-dipped sections were analyzed and photo-graphed by darkfield microscopy using a Nikon MicrophotSA microscope equipped with a 35 mm camera. Low-magnification dark-field photomontages were created bydigitizing the microscopic images at high magnificationwith a Sony XC-77 CCD camera coupled to a Scion LG-3capture board in a PowerMac 7100 computer. Digitizedimages were captured, and overlapping images were placedinto a montage using the Live-Paste function of the publicdomain NIH-Image software package, v. 1.55 (WayneRasband, NIH). Anatomical nomenclature was accordingto the atlas of Paxinos and Watson (1986).

RESULTS

The expression of a1A receptor mRNA showed a veryspecific regional distribution in the rat brain, illustratedand mapped in the film autoradiographs shown in Figure1. Control hybridization with the sense probe generatedonly light and diffuse background signal (Figs. 1P–R, 6B).Background labeling tended to be highest over major fibertracts, but never showed the distinct cellular localizationseen with the antisense riboprobe.

Telencephalon

Olfactory regions and basal forebrain. a1A receptormessage was expressed at very high levels in the olfactorybulb and accessory olfactory nuclei (Fig. 1A), with expres-sion being especially concentrated in the internal granulecell layer of the olfactory bulb proper. No labeling was seenover the olfactory glomeruli. High levels of a1A messageexpression were also seen in the tenia tectae medial to theolfactory nuclei (Fig. 1B,C), extending caudally and dor-sally into the septal region. In the basal forebrain, moder-ate labeling was seen over the vertical nucleus of the

diagonal band, bed nuclei of the stria terminalis, and theventral pallidum, while more intense signal was seen inthe horizontal nucleus of the diagonal band (Fig. 1D). Veryintense hybridization signal was seen more laterally overthe magnocellular preoptic area (Figs. 1E, 6A). The medialand lateral preoptic areas also contained intensely labeledcells, though they were less compact than in the magnocel-lular subregion. These cells extended in a somewhatcontinuous band into and through the anterior lateralhypothalamic area.

Striatum. Little or no specific a1A receptor messageexpression was seen in the neostriatum (Fig. 1C–E), nor inthe ventral extensions of the striatum including the nucleusaccumbens.

Cortex. The neocortex as a whole showed a diffusepattern of moderately dense hybridization, with labeledcells scattered fairly uniformly throughout all corticallayers in all orbital, frontal, parietal, temporal and occipi-tal cortical regions (Figs. 1, 2B, 3). Labeling appeared to beonly slightly more dense and intense in frontal corticalregions as compared to more caudal areas (Fig. 3), andthere were no obvious dorsal-ventral gradients of expres-sion. Neither was there an obvious predominance in anycortical layers. The cingulate cortex and the claustrumwere labeled more heavily than other areas of anteriorcortex (Fig. 1C,D). Piriform cortex was heavily labeledthroughout its rostral-caudal extent, from the level of theolfactory nucleus back to peri-amygdalar levels (Figs.1B–H, 2A, 5).

Hippocampus. The dorsal hippocampus showed a gen-erally modest, though distinct pattern of a1A hybridization(Fig. 1F–J). Individual labeled cells were scattered through-out the CA1 region. A similarly sparse distribution ofsingle cells was seen in the CA3 region and in the dentategyrus, with a1A-expressing cells seen in the pyramidal celllayer as well as the stratum radiatum of the CA fields.Both the CA3 and the DG showed a generally higher levelof hybridization signal than did the CA1. In addition,several intensely labeled cells were seen in the hilus of thedentate gyrus (Fig. 4), and a diffuse band of elevated signalappeared over the granule cell layer. In the ventral hippo-campus, the difference between CA and dentate labelingbecame even more obvious. While the CA fields remainedrather lightly labeled throughout, the granule cell layer ofthe dentate gyrus showed a very intense hybridizationsignal restricted to the caudal and ventral hippocampalregions (Figs. 1I,J, 4).

Diencephalon

Amygdala. At its most anterior level, the medial,basolateral, and lateral amygdalar subnuclei showed mod-erate a1A receptor mRNA expression (Fig. 1F). More cau-dally, the basolateral and lateral amygdala remainedfairly uniformly and moderately labeled, while signal inthe medial and cortical subnuclei were considerably moreintense (Figs. 1G,H, 5). In the central nucleus of theamygdala, only very light labeling was seen in the medialportion. Small clusters of more intensely labeled neuronswere seen dorsal and medial to the central nucleus proper(Fig. 5). The dorsal endopiriform nucleus showed light tomoderate labeling.

Hypothalamus and thalamus. The very dense andintense labeling seen in the preoptic area of the basalforebrain persisted into the anterior lateral hypothalamicarea. The supraoptic nucleus showed moderate a1A mRNA


Fig. 1. A–O: Map of the distribution of a1A adrenergic receptormRNA hybridization signal in rat brain from olfactory bulb (A)through rostral medulla oblongata (N) and thoracic spinal cord (O).The left half of each panel is comprised of a digitized film autoradio-graphic image of brain sections hybridized with the a1A receptor probe,while the right half is a schematic representation indicating the majorbrain structures in that section by cresyl staining, as described in the

text (nomenclature according to Paxinos and Watson, 1986). Numbersin the upper right corner of each panel indicate the correspondingplate numbers in Paxinos and Watson (1986). P–R: A lack of label bythe control sense strand probe in representative forebrain, hindbrainand thoracic spinal cord sections, comparable to the antisense-labeledsections in G,N,O, respectively. See abbreviation list. Scale bar 51 mm.

expression, but very low labeling was seen in themagnocellular neurons of the paraventricular nucleus.Similarly sparse and light signal was seen in the parvocel-lular paraventricular nucleus. A small number of intenselylabeled cells were seen in the arcuate nucleus surroundingthe base of the third ventricle (Fig. 6C) and in the posteriorcomponent of the anterior hypothalamic area (Fig. 1F).The ventromedial hypothalamic nucleus displayed a very

strong signal (Figs. 1F,G, 6D). A loose collection of labeledneurons was seen in the lateral and posterior hypothala-mus. Caudally, these appeared to extend upward to blendwith intensely labeled cells in the ventral zona incerta orsubincertal region. The lateral mammillary nuclei werealso very heavily labeled (Figs. 1H, 6E).

Dorsal to the hypothalamus, strong a1A receptor messageexpression was seen in the zona incerta (Figs. 1F,G, 6D),

Figure 1 (Continued.)




merging ventrally with cells in the lateral and posteriorhypothalamic fields. Very few regions of the thalamusshowed a1A message expression. A small cluster of cells inthe lateral habenula displayed light hybridization signal,

and there was light scattered labeling in the rostralinterstitial nucleus of the medial longitudinal fasciculus.Several large cells exhibiting a1A receptor message expres-sion were seen in the pretectal thalamic nuclei, as were cells



more caudally situated in the mesencephalic nuclei of theoptic tectum.

Brainstem

Midbrain and pons. Many large cells exhibiting veryintense a1A receptor message labeling were scattered

diffusely throughout the tegmental reticular formationextending from mesencephalon through the pons andmedulla (Figs. 1K–N, 8). In the mesencephalon, the nucleiof the optic tectum were sparsely, though distinctly la-beled, as were the pretectal nuclei. The third cranial nervemotor complex showed a high level of a1A receptor message



expression, as did the magnocellular component of the rednucleus (Figs. 1J, 4). Large cells expressing a1A receptormessage were also seen in the interpeduncular nucleus.The pontine nuclei were heavily labeled (Figs. 1J,K, 7A).The pineal gland was also strongly labeled (Fig. 7B).Sparse and moderate signal was observed in the ventralportion of the central gray above the third nerve motornuclei, while a low level of expression was seen in the

dorsal raphe. Moderate expression was seen in the dorsaltegmental nucleus, parabrachial regions and the dorsalnucleus of the lateral lemniscus (Fig. 1L).

Hindbrain. Several areas in the medulla oblongatashowed very intense expression of a1A receptor message,and many individual cells scattered throughout the me-dullary reticular formation displayed very strong hybrid-ization signal (Figs. 1M,N, 8A,B). Labeling over cranial





nerve motor nuclei, especially the fifth and seventh nervemotor nuclei, was particularly intense (Figs. 1M,N, 7C, 8).Neurons in the raphe magnus, raphe pallidus, and medialgigantocellular reticular nucleus were also heavily labeled(Figs. 1N, 7D). The superior and lateral vestibular nucleidisplayed a high level of expression (Figs. 1N, 8B), withlabeled cells extending up into the cerebellar peduncle.Moderate hybridization signal was seen in the ventralcochlear nucleus, the lateral deep cerebellar nuclei, thetwelfth nerve motor nucleus, and the nucleus prepositushypoglossi. No specific labeling was seen in cerebellarcortex, and the central gray region showed generally lowsignal. In the spinal cord, sections from thoracic segmentstwo to four were examined. Strong hybridization of the a1Areceptor probe was seen in the ventral motor areas, withmore moderate labeling in the autonomic region in spinallamina 10 (Fig. 10).

Dual in situ hybridization with ChAT in thefifth and seventh cranial nerve motor nuclei

Results of dual in situ hybridization experiments combin-ing a radioactive probe for ChAT and a non-radioactiveprobe for the a1A receptor showed that the a1A receptormessage is expressed in cholinergic motor neurons of thefifth and seventh nerve motor nuclei (Fig. 9A,C). Essen-tially 100% co-expression of a1A and ChAT mRNA wasobserved in these regions. In contrast, non-cholinergica1A-expressing neurons in other regions of the same sec-tions, including the lateral vestibular nucleus and thenucleus raphe magnus, showed no non-specific silver grainaccumulation (Fig. 9D). Likewise, there was no non-specific labeling when one of the antisense probes wascombined with the other sense probe (Fig. 9B), verifyingthe specificity of the co-localization seen by double-labeling

Fig. 2. In situ hybridization signal for a1A adrenergic receptor mRNA in rat forebrain telencephalicregions. A: Dense labeling in piriform cortex (Pir). B: Moderate and relatively evenly distributed signal inarea Fr 1 of frontal cortex. Dorsal is to the right, and medial is at bottom. Scale bar 5 200 µm.


with the digoxigenin-a1A and 35S-ChAT antisense ribo-probes.

DISCUSSION

At least two a1 adrenergic receptor subtypes, termed a1Aand a1B, have been identified in brain by pharmacologiccriteria (Morrow and Creese, 1986). While anatomicalresolution by pharmacologic differentiation has been onlycoarse at best, it has generally been suggested that the a1Areceptor binding site was most exclusively represented inhippocampus, while the a1B receptor predominated inthalamus, with both binding sites present in cortex (Wil-son and Minneman, 1989). More recently, with molecularcloning of cDNAs coding for the a1 receptors, it has becomeclear that there are at least three a1 receptor subtypes forwhich mRNA are expressed in brain and other tissues. Ithas been well-demonstrated that the cloned a1B receptorcorresponds to the pharmacologic a1B receptor (Cottechiaet al., 1988; Voigt et al., 1990), but there has beenconsiderable confusion regarding the identity and corre-spondence of the a1D and a1A receptor cDNAs to native a1receptor subtypes. Both of these closely-related recombi-nant subtypes show pharmacologic similarity to the nativea1A receptor binding site, and in fact the a1D subtype wasinitially misidentified as the a1A (and later as the a1A/D)

receptor (Lomasney et al., 1991; Schwinn and Lomasney,1992) before being correctly designated as a1D (Perez et al.,1991). The receptor now correctly identified as the a1Asubtype was initially cloned from bovine tissue, and thefact that there was an apparent lack of expression of thismessage in brain and other tissues, in addition to apparentdiscrepancies in its binding characteristics, led Schwinn etal. (Schwinn et al., 1990) to classify this as a novel a1Creceptor subtype. However, with the subsequent cloningand expression of the human and rat homologs of thebovine cDNA (Perez et al., 1994; Price et al., 1994; Rokoshet al., 1994), it became clear that the binding characteris-tics of this receptor more closely matched those of thenative a1A receptor subtype, and it has since been correctlydesignated as the a1A receptor.

One of the important factors that led to the initialmisclassification of the a1A receptor was an apparent lackof expression in brain and other tissues using the bovineclone as a probe (Schwinn et al., 1990). Expression of themRNA encoding this receptor in rat has now been de-scribed in peripheral vascular tissue (Perez et al., 1994),consistent with the known presence of a1A receptor bindingsites. In the present report, we used a riboprobe derivedfrom the rat cDNA, and have now demonstrated anextensive distribution of a1A adrenergic receptor messageexpression throughout the rat brain. The pattern of expres-

Fig. 3. Digitized microscopic images of in situ hybridization signalfor a1A adrenergic receptor mRNA in rat neocortex. A: Labeling infrontal cortex, region Fr 2/3. B: Labeling in parietal cortex, region Par1. C: Labeling in temporal cortex. D: Labeling in occipital cortex. Note

the similarly uniform and even distribution of cells exhibiting signalthroughout the cortical layers in different regions. Numbers in theupper right corner of each panel indicate the corresponding platenumbers in Paxinos and Watson (1986). Scale bar 5 500 µm.


sion for this receptor shows a distinct regional specificity,with several areas showing very intense hybridizationsignal: olfactory bulb, tenia tectae, horizontal diagonalband/magnocellular preoptic area, zona incerta, ventrome-dial and lateral hypothalamus, ventral dentate gyrus,piriform cortex, lateral mammillary nuclei, medial andcortical amygdala, magnocellular red nuclei, pontine nu-clei, superior and lateral vestibular nuclei, several cranialnerve and spinal motor nuclei, and brainstem reticularnuclei. Other areas showing a more moderate level ofexpression include the neocortex, claustrum, hippocam-pus, vertical diagonal band, bed nuclei of the stria termina-lis, lateral amygdala, central gray, raphe nuclei, ventralcochlear nuclei, and deep cerebellar nuclei.

The probe used in this study corresponds to nt 858-1410of the rat a1A receptor cDNA (Perez et al., 1994), encodingmRNA from the end of the third cytoplasmic loop regionthrough the end of the message. This region shows onlyapproximately 50% homology with both the a1B and a1DcDNA sequences, and considerably less outside the trans-membrane regions. In addition, as considered in moredetail below, the pattern of expression observed in thepresent study is quite distinct from that reported for eitherof these other closely related adrenergic receptor subtypes,further attesting to the selectivity of the labeling, espe-cially under the high stringency conditions used in thecurrent experiment.

This distribution of a1A receptor message is generallyconsistent with results from previous autoradiographicstudies showing non-selective a1 receptor binding sites inneocortex, olfactory bulb, thalamus, hippocampus andbrainstem motor nuclei (Jones et al., 1985; Young andKuhar, 1980). Subregions of the amygdala, especially thelateral and medial amygdala, show a pattern of a1 receptorbinding with a distribution very similar to that of the a1Areceptor mRNA (Flugge et al., 1994), while more moderatebinding has been demonstrated in hypothalamus (Cum-mings and Seybold, 1988). The present description of a1Areceptor message expression is also consistent with themore coarse and limited reports of a1A-like binding incortex, dentate gyrus and brainstem (Gross et al., 1989;Laporte et al., 1991), though it is likely that the a1Dadrenergic receptor may contribute in varying proportionsto the a1A-like binding activity reported in these differentbrain regions.

Functional implications of a1A receptorlocalization compared to the a1B

and a1D subtypes

The distribution of a1A receptor message expressiondescribed herein is distinct from that of both of the twoother a1 adrenergic receptor subtypes, a1B and a1D, asdescribed previously (McCune et al., 1993; Pieribone et al.,

Fig. 4. Digitized photomontage of a1A receptor mRNA hybridiza-tion signal in rat caudal hippocampus. The signal in the dentate gyrus(DG) was much more intense in the ventral region (DGv). Note thegenerally lighter labeling in the CA1 and CA3 regions relative to the

dentate gyrus, and the labeling in scattered cells of the hilar region.Note also the labeling seen in the 3rd motor nucleus (3) and themagnocellular subdivision of the red nucleus (RMC). CA1–3, fields ofAmmon’s horn. Scale bar 5 1mm.


1994). Of the three a1 receptor subtypes, the a1B receptorappears to be the most restricted in its distribution, beinglimited primarily to the thalamus, the middle layers ofneocortex, brainstem motor nuclei, and the raphe nuclei(McCune et al., 1993; Pieribone et al., 1994). In contrast,the a1A receptor (present results) and a1D receptor mes-sages appear to be much more highly and widely expressedthroughout the rat brain (McCune et al., 1993; Pieribone etal., 1994;A. Williams and D. Morilak, unpublished observa-tions). In many regions, these receptor subtypes showoverlapping or complementary expression, while in otherareas the expression appears more exclusive for onesubtype relative to the others.

All three adrenergic receptor subtypes are representedin the neocortex in a moderately dense and somewhatdiffuse fashion. Noradrenergic activation of a1 receptorshas been demonstrated to facilitate sensory-evoked electro-physiological responses in somatosensory and visual corti-cal neurons (Waterhouse et al., 1981, 1983, 1990). Activa-tion of cortical a1 receptors has also been shown topotentiate the release and post-synaptic effects of othercortical neurotransmitters, including GABA, acetylcholineand VIP (Ferraro et al., 1993; Magistretti and Schorderet,1985; Waterhouse et al., 1981). Any of the three a1 receptorsubtypes potentially could participate in these modulatoryeffects on cortical cellular activity and transmission. At amore behavioral level, the widespread cortical expressionof a1 receptors is also most likely responsible for theactivation of cortical EEG and the behaviorally arousingeffects of a1 receptor stimulation, and conversely for thesedative effects of a1 receptor blockade (Guo et al., 1991;Hilakivi and Leppavuori, 1984; Hilakivi-Clarke et al.,1991). While specific roles for the different a1 receptor

subtypes in modulating neocortical activity cannot beinferred from their similarly diffuse distribution, in themore primitive piriform cortex there is an apparentlyexclusive expression of the a1A receptor message as com-pared to the a1B and a1D receptor subtypes (McCune et al.,1993; Pieribone et al., 1994). Norepinephrine convergeswith other monoaminergic afferents in piriform cortex toamplify IPSPs in pyramidal cells by activating interneu-rons (Gellman and Aghajanian, 1993). Given the pattern ofa1 receptor subtype mRNA expression in piriform cortex,this effect is apparently mediated solely by a1A receptors.

Unlike the similar and diffuse labeling seen in theneocortex, the three a1 receptor subtypes show quitedistinct relative patterns of mRNA expression in thehippocampus, where a1 receptor activation has been shownto amplify evoked population spike activity in the dentategyrus (Winson and Dahl, 1985). While the a1B receptordoes not appear to be expressed at all in hippocampus, thea1D receptor message is present at high levels in all regions(McCune et al., 1993; Petit et al., 1995; Pieribone et al.,1994; Williams et al., 1997). In contrast, the a1A receptormessage is also expressed throughout the hippocampus,but the level of expression is relatively low and limited tofew cells, except in the ventral hippocampus, where veryintense expression is seen selectively in the ventral den-tate gyrus. The ventral hippocampus has been shown to bean important component in the feedback regulation ofstress-induced hypothalamo-pituitary-adrenal activationand subsequent glucocorticoid secretion, an effect thoughtto be mediated by glucocorticoid receptors in the ventralhippocampus (Herman et al., 1989). Noradrenergic inner-vation of the hippocampus is derived from the pontine

Fig. 5. Digitized photomontage of a1A receptor mRNA hybridiza-tion signal in the amygdala. Labeling was most intense in the medial(Me) and cortical (PLCo) amygdalar subnuclei, and more moderate inthe lateral (La) and basolateral (BL) subnuclei. Signal was generally

very low in the central nucleus (CeM and CeL), with small clusters oflabeled cells located dorsal and medial to the central nucleus. Pir,Piriform cortex. Scale bar 5 500 µm.


Fig. 6. In situ hybridization of a1A adrenergic receptor mRNA indiencephalic regions of rat brain. A: Very intense labelling of largecells in the magnocellular preoptic area. B: A section adjacent to thatin A, showing the same brain region after hybridization with thecontrol sense probe. Note the lack of specific signal. C: Labeled cells in

the hypothalamic arcuate nucleus surrounding the base of the thirdventricle (v). D: Strong hybridization signal seen in cells of the medialzona incerta (ZI) and ventromedial hypothalamic nucleus (VMH).v, Third ventricle. E: Very high expression in the lateral mammillarynucleus (LM). Scale bar 5 250 µm in A,B,D,E, and 100 µm in C.


locus coeruleus, and these noradrenergic neurons havebeen shown to be activated by a variety of stressors(Jacobs, 1986; Jacobs et al., 1991). Thus, it is possible thatthere may be a specific role for a1A adrenergic receptors inmodulating the feedback effects of the ventral hippocam-pus on the HPA axis during stress. In addition, a1Areceptors may also modulate the hormonal stress responseby influencing converging afferents to the hypothalamicHPA axis arising from the amygdala, the arcuate nucleus,and the preoptic area.

The a1A receptor also appears to be selectively expressedin a number of specific subnuclei of the hypothalamus,where there is relatively little expression of either the a1Bor a1D receptor subtypes (Pieribone et al., 1994). In thehypothalamus, the a1A receptor mRNA is expressed athigh levels in the magnocellular preoptic area, supraopticnucleus, arcuate nucleus, ventromedial hypothalamus,lateral hypothalamic area, and lateral mamillary nuclei.Expression in the arcuate and ventromedial nuclei are

especially intriguing, as these are areas within which a1adrenergic receptors might interact with neuropeptidetransmitters to exert modulatory influences on neuroendo-crine and feeding-related processes (Parker and Crowley,1993). For example, the potently orexigenic neuropeptideY (NPY) is co-localized with norepinephrine in ascendingafferents to the hypothalamus arising from the ventrolat-eral medulla and locus coeruleus (Sawchenko et al., 1985).NPY is also highly concentrated in the arcuate, ventrome-dial and paraventricular hypothalamus. Thus, noradrener-gic activation of a1A receptors in these regions may influ-ence NPY-induced feeding either directly by altering therelease of NPY from neurons in the hypothalamus, or bymodulating the post-synaptic effects of co-released NPY oncommon hypothalamic target neurons, as has been demon-strated in peripheral vascular tissue (Selbie et al., 1995).

There appears to be considerable overlap of the three a1receptor subtypes in the hindbrain. Along with the a1Areceptor mRNA described in the present study, the a1B and

Fig. 7. Expression of a1A adrenergic receptor mRNA in regions of the midbrain and brainstem. A: Intenselabeling in the pontine nuclei (Pn). B: Strong hybridization signal in the pineal gland. C: Large, intenselylabeled neurons in the facial motor nucleus (7). D: a1A mRNA expression in the nucleus raphe magnus(RMg)on the ventral midline, overlying the pyramidal tracts. Scale bar 5 250 µm.


Fig. 8. Digitized microscopic images of a1A adrenergic receptormRNA expression in the pons-medulla. A: Labeling seen at the level ofthe trigeminal motor nucleus (Mo5), in which a very high level of a1AmRNA expression was observed. Hybridization signal was also promi-nent in large cells scattered throughout the pontine reticular nucleus

(PnC). B: Labeling in the rostral medulla oblongata, at the level of thefacial motor nucleus (7). Very intense labeling was seen over large cellsin the facial nucleus, in the superior and lateral vestibular nuclei(SuVe, LVe), and in large cells throughout the gigantocelluler reticularformation (Gi, GiA). Scale bar 5 1 mm.

a1D receptor mRNAs are also expressed at high levels inbrainstem reticular nuclei, and are especially dense incranial nerve motor nuclei (Pieribone et al., 1994). Dual insitu hybridization in the present study verified that the a1Amessage is expressed in cholinergic motor neurons in theseregions. Norepinephrine, acting upon a1 receptors, hasbeen shown to facilitate evoked activation of motor neu-rons in the seventh nerve nucleus (VanderMaelen andAghajanian, 1980). Likewise, norepinephrine facilitateselicitation of the masseteric jaw closure reflex throughactivation of a1 adrenergic receptors in the fifth nerve

motor nucleus (Morilak and Jacobs, 1985), and selectivelesioning has suggested that these receptors are localizedon the motor neurons (Shao and Sutin, 1991). In freelymoving animals, masseteric reflex facilitation by a1 adren-ergic receptors occurs in response to mildly stressfulstimuli previously shown to increase noradrenergic cellu-lar activity (Stafford and Jacobs, 1990a, b), again suggest-ing a state-dependent modulatory role for a1 receptors instress. Adrenergic facilitation of spinal motor neuronresponses (White and Neuman, 1983) may also be attrib-uted to any or all of the a1 receptor subtypes, as all three

Fig. 9. Dual in situ hybridization for the a1A adrenergic receptormRNA and choline acetyltransferase (ChAT) mRNA in cholinergicmotor neurons of the fifth and seventh cranial nerve motor nuclei. Thedigoxigenin-labeled a1A mRNA was visualized by a colorimetric alka-line-phosphatase reaction, seen as a solid purple precipitate in thecytoplasm of positive cells. The [35S]-labeled ChAT mRNA was detectedautoradiographically, visualized as black silver grains deposited in theemulsion layer above the cytoplasm of positive cells. Digoxigenin-labeled cells are slightly out of focus, as the plane of focus was on theemulsion layer above the tissue to visualize silver grains. A: Motorneurons in the fifth motor nucleus displaying hybridization signal for

both the a1A adrenergic receptor and ChAT, indicating colocalization.B: A section adjacent to that in A, hybridized with the a1A receptorantisense probe (purple cells) and the control sense probe for ChAT.The lack of silver grains in a1A-positive cells demonstrates thespecificity of the dual-labeling procedure. C: Double-labeled neuronsin the seventh motor nucleus. D: A control cell in the lateral vestibularnucleus (LVe) expressing a1A adrenergic receptor mRNA, indicated bythe purple reaction product. This section was also hybridized with the[35S]-labeled ChAT probe, and the lack of silver grain deposition in theLVe, where there are no cholinergic neurons, further illustrates thespecificity of the dual-labeling procedure. Scale bar 5 50 µm.


are amply represented in the ventral motor regions of thespinal cord.

CONCLUSION

The effects of noradrenergic neurotransmission in thebrain have been described as neuromodulatory in nature,and many of these modulatory effects have been attributedto a1 adrenergic receptor activation. However, it hasgenerally not been possible to reliably differentiate thephysiological effects of the known a1 receptor subtypesusing pharmacological means, especially the a1A and a1Dreceptor subtypes. Thus, one step toward understandingthe specificity of their actions and the physiological con-texts in which these receptors might differ is to delineatetheir differential distribution and expression in areas ofthe brain in which a1-mediated modulatory effects havebeen observed. In the present study, we describe thedistribution of a1A adrenergic receptor messenger RNAexpression in rat brain. Expression of a1A receptor mes-sage was overlapping or complementary with expression ofthe a1B or a1D receptor subtypes in olfactory bulb, neocor-tex, hippocampus, amygdala, brainstem reticular nuclei,and brainstem motor nuclei. The a1A receptor message isexpressed predominantly or exclusively in piriform cortex,several subnuclei of the hypothalamus, ventral hippocam-pus, zona incerta, and pontine nuclei. In contrast, previousdescriptions suggest that the a1D receptor shows predomi-nant expression in the CA fields of the hippocampus,especially in the dorsal hippocampus, reticular thalamicnucleus, and inferior olive, while the a1B receptor predomi-nates in the thalamus and dorsal raphe (Pieribone et al.,1994). Modulatory effects of a1 receptor activation havebeen described in all these regions. Thus, additionalbiochemical and molecular studies addressing potentialdifferences in signal transduction (Sulpizio and Hieble,1991; Tsujimoto et al., 1989) or differential regulation ofthe three a1 adrenergic receptor subtypes by drugs, hor-mones, or complex physiological stimuli (Blendy et al.,1990, 1991; Grimm et al., 1992; Petitti et al., 1992) will benecessary in order to fully understand the functionalsignificance of their specific regional localization in mediat-ing noradrenergic modulation in the brain.

ACKNOWLEDGMENTS

We thank Dr. Dianne Perez, Cleveland Clinic Founda-tion, for kindly providing the cDNA plasmid pMT28-Ra1A,and Dr. Christine Gall, University of California, Irvine, forproviding the cDNA plasmid for ChAT. We also thank Ms.Amelia Williams for excellent technical assistance.

LITERATURE CITED

Aghajanian, G.K., and M.A. Rogawski (1983) The physiological role ofa-adrenoceptors in the CNS: New concepts from single-cell studies.Trends Pharmacol. Sci. 4:315–317.

Blendy, J.A., L.J. Grimm, D.C. Perry, L. West-Johnstrud, and K.J. Kellar(1990) Electroconvulsive shock differentially increases binding to al-pha-1 adrenergic receptor subtypes in discrete regions of rat brain. J.Neurosci. 10:2580–2586.

Blendy, J.A., D.C. Perry, L.A. Pabreza, and K.J. Kellar (1991) Electroconvul-sive shock increases a1b- but not a1a-adrenoceptor binding sites in ratcerebral cortex. J. Neurochem. 57:1548–1555.

Cottechia, S., D.A. Schwinn, R.R. Randall, R.J. Lefkowitz, M.G. Caron, andB.K. Kobilka (1988) Molecular cloning and expression of the cDNA for

the hamster a1-adrenergic receptor. Proc. Nat. Acad. Sci. USA 85:7159–7163.

Cummings, S., and V. Seybold (1988) Relationship of alpha-1 and alpha-2-adrenergic-binding sites to regions of the paraventricular nucleus of thehypothalamus containing corticotropin-releasing factor and vasopres-sin neurons. Neuroendocrinology 47:523–532.

Ferraro, L., S. Tanganelli, G. Calo, T. Antonelli, A. Fabrizi, N. Acciarri, C.Bianchi, L. Beani, and M. Simonato (1993) Noradrenergic modulationof g-aminobutyric acid outflow from the human cerebral cortex. BrainRes. 629:103–108.

Flugge, G., O. Ahrens, and E. Fuchs (1994) Monoamine receptors in theamygdaloid complex of the tree shrew (Tupaia belangeri). J. Comp.Neurol. 343:597–608.

Gellman, R.L., and G.K. Aghajanian (1993) Pyramidal cells in piriformcortex receive a convergence of inputs from monoamine activatedGABAergic interneurons. Brain Res. 600:63–73.

Grimm, L.J., J.A. Blendy, K.J. Kellar, and D.C. Perry (1992) Chronicreserpine administration selectively up-regulates b1- and a1b-adrener-gic receptors in rat brain: An autoradiographic study. Neuroscience47:77–86.

Gross, G., G. Hanft, and H.M. Mehdorn (1989) Demonstration of a1A- anda1B-adrenoceptor binding sites in human brain tissue. Eur. J. Pharma-col. 169:325–328.

Guo, T.-Z., J. Tinklenberg, R. Oliker, and M. Maze (1991) Central a1-adrenoceptor stimulation functionally antagonizes the hypnotic re-sponse to dexmedetomidine, an a2-adrenoceptor agonist. Anesthesiol-ogy 75:252–256.

Herman, J.P., M.K.H. Schafer, E.A. Young, R. Thompson, J. Douglass, H.Akil, and S.J. Watson (1989) Evidence for hippocampal regulation ofneuroendocrine neurons of the hypothalamo-pituitary-adrenocorticalaxis. J. Neurosci. 9:3072–3082.

Hieble, J.P., D.B. Bylund, D.E. Clarke, D.C. Eikenburg, S.Z. Langer, R.J.Lefkowitz, K.P. Minneman, and R.R. Ruffolo (1995) International unionof pharmacology X. Recommendation for nomenclature of a1-adrenocep-tors: Consensus update. Pharmacol. Revs. 47:267–271.

Hilakivi, I., and A. Leppavuori (1984) Effects of methoxamine, an alpha-1adrenoceptor agonist, and prazosin, an alpha-1 antagonist, on thestages of the sleep-waking cycle in the cat. Acta Physiol. Scand.120:363–372.

Hilakivi-Clarke, L.A., J. Turkka, R.G. Lister, and M. Linnoila (1991) Effectsof early postnatal handling on brain b-adrenoceptors and behavior intests related to stress. Brain Res. 542:286–292.

Jacobs, B.L. (1986) Single unit activity of locus coeruleus neurons inbehaving animals. Prog. Neurobiol. 27:183–194.

Jacobs, B.L., E.D. Abercrombie, C.A. Fornal, E.S. Levine, D.A. Morilak, andI.L. Stafford (1991) Single-unit and physiological analyses of brainnorepinephrine function in behaving animals. Prog. Brain Res. 88:159–165.

Jones, L.S., L.L. Gauger, and J.N. Davis (1985) Anatomy of brain alpha1-adrenergic receptors: In vitro autoradiography with [125I]-HEAT. J.Comp. Neurol. 231:190–208.

Laporte, A.-M., L.E. Schechter, F.J. Bolanos, D. Verge, M. Hamon, and H.Gozlan (1991) [3H]5-Methyl-urapidil labels 5-HT1A receptors and a1-adrenoceptors in the rat CNS. In vitro binding and autoradiographicstudies. Eur. J. Pharmacol. 198:59–67.

Lauterborn, J.C., P.J. Isackson, R. Montalvo, and C.M. Gall (1993) In situhybridization localization of choline acetyltransferase mRNA in adultrat brain and spinal cord. Mol. Brain Res. 17:59–69.

Lomasney, J.W., S. Cotecchia, W. Lorenz, W.-Y. Leung, D.A. Schwinn, T.L.Yang-Feng, M. Brownstein, R.J. Lefkowitz, and M.G. Caron (1991)Molecular cloning and expression of the cDNA for the a1A-adrenergicreceptor, the gene for which is located on human chromosome 5. J. Biol.Chem. 266:6365–6369.

Magistretti, P.J., and M. Schorderet (1985) Norepinephrine and histaminepotentiate the increases in cyclic adenosine 38:58-monophosphate elic-ited by vasoactive intestinal polypeptide in mouse cerebral corticalslices: Mediation by a1-adrenergic and H1-histaminergic receptors. J.Neurosci. 5:362–368.

McCune, S.K., M.M. Voigt, and J.M. Hill (1993) Expression of multiplealpha adrenergic receptor subtype messenger RNAs in the adult ratbrain. Neuroscience 57:143–151.

Morilak, D.A., and B.L. Jacobs (1985) Noradrenergic modulation of sensori-motor processes in intact rats: The masseteric reflex as a model system.J. Neurosci. 5:1300–1306.


Morrow, A.L., and I. Creese (1986) Characterization of a1-adrenergicreceptor subtypes in rat brain: A reevaluation of [3H]WB4101 and[3H]prazosin binding. Mol. Pharmacol. 29:321–330.

Parker, S.L., and W.R. Crowley (1993) Central stimulation of oxytocinrelease in the lactating rat: Interaction of neuropeptide Y with a-1-adrenergic mechanisms. Endocrinology 132:658–666.

Paxinos, G., and C. Watson (1986) The Rat Brain in Stereotaxic Coordi-nates, 2nd Edition. San Diego: Academic Press.

Perez, D.M., M.T. Piascik, and R.M. Graham (1991) Solution-phase libraryscreening for the identification of rare clones: Isolation of an a1D-adrenergic receptor cDNA. Mol. Pharmacol. 40:876–883.

Perez, D.M., M.T. Piascik, N. Malik, R. Gaivin, and R.M. Graham (1994)Cloning, expression, and tissue distribution of the rat homolog of thebovine a1C-adrenergic receptor provide evidence for its classification asthe a1A subtype. Mol. Pharmacol. 46:823–831.

Petit, R.L., A.M. Williams, and D.A. Morilak (1995) Colocalization of a1A/Dadrenoreceptor mRNA with type I and type II glucocorticoid receptormRNA in rat hippocampus using double in situ hybridization. Soc.Neurosci. Abstr. 21:1614.

Petitti, N., G.B. Karkanias, and A.M. Etgen (1992) Estradiol selectivelyregulates a1B-noradrenergic receptors in the hypothalamus and preop-tic area. J. Neurosci. 12:3869–3876.

Pieribone, V.A., A.P. Nicholas, A. Dagerlind, and T. Hokfelt (1994) Distribu-tion of a1 adrenoceptors in rat brain revealed by in situ hybridizationexperiments utilizing subtype-specific probes. J. Neurosci. 14:4252–4268.

Price, D.T., R.S. Chari, D.E. Berkowitz, W.C. Meyers, and D.A. Schwinn(1994) Expression of a1-adrenergic receptor subtype mRNA in rattissues and human SK-N-MC neuronal cells: Implications for a1-adrenergic receptor subtype classification. Mol. Pharmacol. 46:221–226.

Rokosh, D.G., B.A. Bailey, A.F.R. Stewart, L.R. Karns, C.S. Long, and P.C.Simpson (1994) Distribution of a1C-adrenergic receptor mRNA in adultrat tissues by RNase protection assay and comparison with a1B anda1D. Biochem. Biophys. Res. Comm. 200:1177–1184.

Sawchenko, P.E., L.W. Swanson, R. Grzanna, P.R.C. Howe, S.R. Bloom, andJ.M. Polak (1985) Colocalization of neuropeptide Y immunoreactivity inbrainstem catecholaminergic neurons that project to the paraventricu-lar nucleus of the hypothalamus. J. Comp. Neurol. 241:138–153.

Schwinn, D.A., and J.W. Lomasney (1992) Pharmacologic characterizationof cloned a1-adrenoceptor subtypes: Selective antagonists suggest theexistence of a fourth subtype. Eur. J. Pharmacol. 227:433–436.

Schwinn, D.A., J.W. Lomasney, W. Lorenz, P.J. Szklut, R.T. Fremeau, T.L.Yang-Feng, M.G. Caron, R.J. Lefkowitz, and S. Cotecchia (1990)Molecular cloning and expression of the cDNA for a novel a1-adrenergicreceptor subtype. J. Biol. Chem. 265:8183–8189.

Selbie, L.A., K. Darby, C. Schmitz-Peiffer, C.L. Browne, H. Herzog, J. Shine,and T.J. Biden (1995) Synergistic interaction of Y1-neuropeptide Y anda1b-adrenergic receptors in the regulation of phospholipase C, proteinkinase C, and arachidonic acid production. J. Biol. Chem. 270:11789–11796.

Shao, Y., and J. Sutin (1991) Noradrenergic facilitation of motor neurons:Localization of adrenergic receptors in neurons and nonneuronal cellsin the trigeminal motor nucleus. Exp. Neurol. 114:216–227.

Stafford, I.L., and B.L. Jacobs (1990a) Noradrenergic modulation of themasseteric reflex in behaving cats. I. Pharmacological studies. J.Neurosci. 10:91–98.

Stafford, I.L., and B.L. Jacobs (1990b) Noradrenergic modulation of themasseteric reflex in behaving cats. II. Physiological studies. J. Neuro-sci. 10:99–107.

Sulpizio, A., and J.P. Hieble (1991) Lack of a pharmacological distinctionbetween alpha-1 adrenoceptors mediating intracellular calcium-dependent and independent contractions to sympathetic nerve stimula-tion in the perfused rat caudal artery. J. Pharmacol. Exper. Therap.257:1045–1052.

Tsujimoto, G., A. Tsujimoto, E. Suzuki, and K. Hashimoto (1989) Glycogenphosphorylase activation by two different a1-adrenergic receptor sub-types: Methoxamine selectively stimulates a putative a1-adrenergicreceptor subtype (a1a) that couples with Ca21 influx. Mol. Pharmacol.36:166–176.

VanderMaelen, C.P., and G.K. Aghajanian (1980) Intracellular studiesshowing modulation of facial motoneurone excitability by serotonin.Nature 287:346–347.

Voigt, M.M., J. Kispert, and H. Chin (1990) Sequence of a rat brain cDNAencoding an alpha-1B adrenergic receptor. Nucleic Acids Res. 18:1053.

Waterhouse, B.D., S.A. Azizi, R.A. Burne, and D.J. Woodward (1983)Interactions of norepinephrine and serotonin with visually evokedresponses of simple and complex cells in area 17 of rat cortex. Soc.Neurosci. Abstr. 9:1001.

Waterhouse, B.D., S.A. Azizi, R.A. Burne, and D.J. Woodward (1990)Modulation of rat cortical area 17 neuronal responses to moving visualstimuli during norepinephrine and serotonin microiontophoresis. BrainRes. 514:276–292.

Waterhouse, B.D., H.C. Moises, and D.J. Woodward (1981) Alpha-receptormediated facilitation of somatosensory cortical neuronal responses toexcitatory synaptic inputs and iontophoretically applied acetylcholine.Neuropharmacol. 20:907–920.

Waterhouse, B.D., F.M. Sessler, W. Liu, and C.-S. Lin (1991) Secondmessenger-mediated actions of norepinephrine on target neurons incentral circuits: A new perspective on intracellular mechanisms andfunctional consequences. Prog. Brain Res. 88:351–362.

White, S.R., and R.S. Neuman (1983) Pharmacological antagonism offacilitatory but not inhibitory effects of serotonin and norepinephrineon excitability of spinal motoneurones. Neuropharmacol. 22:489–494.

Williams, A.M., M.L.D. Nguyen, and D.A. Morilak (1997) Co-localization ofa1D adrenergic receptor mRNA with mineralocorticoid and glucocorti-coid receptor mRNA in rat hippocampus. J. Neuroendocrinol. 9:113–119.

Wilson, K.M., and K.P. Minneman (1989) Regional variations in a1-adrenergic receptor subtypes in rat brain. J. Neurochem. 53:1782–1786.

Winson, J., and D. Dahl (1985) Action of norepinephrine in the dentategyrus. II.Iontophoretic studies. Exp. Brain Res. 59:497–506.

Young, W.S., and M.J. Kuhar (1980) Noradrenergic a1 and a2 receptors:Light microscopic autoradiographic localization. Proc. Natl. Acad. Sci.USA 77:1696–1700.


Documents

Distribution of ?1A adrenergic receptor m RNA in the rat brain visualized by in situ hybridization