Introduction tigation was to determine whether chronic psychosocial

Brain Research 880 2000 147–158 www.elsevier.com locate bres Research report Regulation of a -adrenoceptor expression by chronic stress in 2A neurons of the brain stem a a b a , ¨ Heiko Meyer , Monika Palchaudhuri , Mika Scheinin , Gabriele Flugge a ¨ Division of Neurobiology , German Primate Center, Kellnerweg 4, D-37077 Gottingen, Germany b Department of Pharmacology and Clinical Pharmacology , University of Turku, FIN-20520 Turku, Finland Accepted 2 August 2000 Abstract a -Adrenoceptors are supposed to be important regulatory elements in responses to stress. Previous receptor binding studies in male 2 tree shrews have shown that chronic psychosocial stress down-regulates binding sites for a -adrenergic ligands in several brain stem 2 nuclei. The aim of the present study was to quantify effects of chronic subordination stress on expression of the a -adrenoceptor subtype 2 A gene in identified neurons of the brain stem. We partially cloned the a -adrenoceptor cDNA of the tree shrew 1.22 kb and localized 2A 35 receptor RNA expression in brain stem neurons by in situ hybridization using a S-labeled cRNA probe 1.06 kb. To identify neurons expressing receptor mRNA, brain sections were first immunocytochemically stained with antibodies against tyrosine hydroxylase, phenylethanolamine-N-methyltransferase, or glutamate, and then processed for in situ hybridization. Furthermore, expression of receptor-specific RNA was quantified in single neurons of animals which had been psychosocially stressed during 4 weeks and in unstressed controls. We found strong in situ hybridization in the noradrenergic neurons of the locus coeruleus, but only weak labeling of A2 neurons in the solitary tract nucleus and no labeling of A1 neurons in the caudal ventrolateral medulla. Adrenergic neurons in the solitary tract nucleus group C2 did not express the a -adrenoceptor, and C1 neurons in the rostral ventrolateral medulla showed only a 2A minor labeling by the in situ probe. In contrast, large glutamatergic neurons in the lateral reticular nucleus were strongly labeled by the probe. Chronic psychosocial stress reduced a -adrenoceptor RNA expression in locus coeruleus neurons 224.0, in solitary tract 2A neurons 231.0, and in neurons of the lateral reticular nucleus 218.8. These findings show that stress not only decreases the expression of the a -adrenergic autoreceptor in the locus coeruleus but also of a -heteroreceptors in glutamatergic neurons.  2000 2A 2A Elsevier Science B.V. All rights reserved. Theme : Neural basis of behaviour Topic : Stress Keywords : a -Adrenoceptor; Chronic stress; Tree shrew; Locus coeruleus; Lateral reticular nucleus 2A

1. Introduction tigation was to determine whether chronic psychosocial

stress alters expression of the a -adrenoceptor subtype A. 2 Previous work from our group has shown that chronic Several pieces of experimental evidence indicate that this subordination stress down-regulates a -adrenoceptors in subtype is important in the locus coeruleus LC, where it 2 selected brain nuclei [6]. While these receptor binding regulates neuronal activity and therefore also noradrenaline studies demonstrated a decrease in numbers of binding release in the projection areas of the LC [2,4,17,32]. sites for radioligands, the experiments could not show The distribution of a -adrenoceptor expression has 2A whether the effects were due to reduced expression of been studied in rat and mouse brain [25,33,35,40]. Al- receptor genes and which of the a -adrenoceptor subtypes though it was demonstrated that the receptor is strongly 2 was affected. Therefore, the aim of the present inves- expressed in the noradrenergic LC neurons, it is not entirely clear whether also other noradrenergic or adren- ergic cells express it. Electron microscopic studies re- Corresponding author. Tel.: 149-551-3851-133: fax: 149-551-3851- vealed that most of the a -adrenoceptor immunoreactive 228. 2A ¨ E-mail address : gfluggewww.dpz.gwdg.de G. Flugge. structures in the rostral ventrolateral medulla do not 0006-8993 00 – see front matter  2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 7 8 7 - 6 148 H contain tyrosine hydroxylase, indicating that they are not 2.2. a -Adrenoceptor cloning and cRNA probe 2 a noradrenergic [23]. We therefore identified neurons in the tree shrew brain stem expressing the a -adrenoceptor by PolyA1-RNA was extracted from the tree shrew brain 2A combining in situ hybridization and immunocytochemical stem with the Micro Fast Tract kit Invitrogen, Leek, techniques. Expression of mRNA for the receptor was Netherlands and cDNA was generated using the cDNA- quantified in neurons of male tree shrews that had been Cycle kit Invitrogen. Published a -AR cDNA se- 2A psychosocially stressed for four weeks and in unstressed quences of human, rat and mouse [15,16,18], EMBL controls. GenBank data bank EMBL, Heidelberg, Germany and Male tree shrews Tupaia belangeri provide an animal IntelliGenetics Mountain View, CA, USA were used to model to study the effects of chronic psychosocial stress design two oligonucleotide primers from the conserved [9]. When two males are kept together in one cage, there elements of the coding regions of the a -adrenoceptor 2A are short social encounters whereupon a clear dominant- gene size of coding region, 1.35 kb. The sequence 59- subordinate hierarchy is established. During such periods CGCGGCGGAAATCGTGGTTGAAGAT-3 served as re- of stress, the subordinate shows persistent activation of the verse, and 59-CTGCAGGTGACGCTGACGCTGGTGT-3 sympathetic–adrenomedullary system and the hypo- as forward primer KEBO Lab, Espoo, Finland. The PCR thalamic–pituitary–adrenal HPA axis. In the brains of was performed at 96, 67 and 728C, each for 60 s 35 subordinates, the noradrenergic system is chronically acti- cycles in the presence of 10 mM Tris–HCl pH 8.8, 50 vated, as reflected by a high noradrenaline turnover rate mM KCl, 0.5 mM MgCl , 0.1 Triton X-100 and 200 mM 2 [30]. dNTP-mix with 100 ng cDNA, 2 units DynaZyme DNA In the present study, a -adrenoceptor expression in the polymerase Finnzymes, Espoo, Finland, and 50 pmol of 2A tree shrew brain stem was visualized by in situ hybridiza- each primer. The amplification reaction was terminated by tion. To obtain probes for the experiments, a 1.22-kb primer extension at 728C for 25 min. Three PCR-products cDNA-fragment of the tree shrew a -adrenoceptor was of approximately 1.2 kb were subcloned in pGEM-T 2A 35 cloned using RT-PCR techniques. A [ S]UTP labeled Promega, Madison, WI, USA, and characterized by run-off cRNA transcript was generated to visualize the manual sequence analysis using the Sequenase 2.0 kit distribution of receptor mRNA on autoradiography films. Pharmacia, Biotech, Uppsala, Sweden. For cRNA pro- In brain sections coated with photographic emulsion, the duction, the plasmid was cut with BglII Pharmacia receptor mRNA expression was visualized in single neu- Biotech, Uppsala, Sweden to generate a 1057-nucleotide rons that were identified with antibodies against catechol- fragment complementary to positions 238–1219 of the amine-synthesizing enzymes and against glutamate. For cloned cDNA fragment and 75 nucleotides of the vector semi-quantitative analysis of receptor expression in chroni- antisense or NotI Promega; sense. The linearized cDNA cally stressed animals and in controls, silver grains were was in vitro-transcribed with the Riboprobe in vitro-tran- counted over single neurons. scription system Promega, using T7-polymerase to gener- ate the antisense and SP6-polymerase to generate the sense 35 probe in the presence of [ S]UTP 250 mCi; ICN Pharma- ceuticals, Costa Mesa, CA. The probes were purified with

2. Materials and methods S400 HR MicroSpin columns Pharmacia. Labeling ef-