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

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-

ficiency was analyzed in a scintillation counter and integri- 2.1. Animal experiments ty was checked on a 6 polyacrylamide gel. Male tree shrews Tupaia belangeri from the breeding 2.3. Immunocytochemistry colony at the German Primate Center were used [8]. All experiments were conducted in accordance with the Euro- To identify neurons in the brain stem, paraffin sections pean Communities Council Directive of 24 November 5 mm from brains were used that had been perfusion 1986 86 EEC and approved by the Government of fixed with 4 paraformaldehyde PFA according to Lower Saxony, Germany. Prior to the experiment, all standard procedures [22]. After removing the paraffin, males passed a 10-day control period without stress. sections were processed for immunocytochemistry, and Subordinates stress group; n54 were then submitted to binding of the primary antibody was visualized by the psychosocial stress for 28 days as described before [9]. peroxidase–anti-peroxidase technique as described [7,22]. Unstressed control animals were kept in a separate room The following primary antibodies were used: anti-tyrosine n54. The stress level of the subordinates was determined hydroxylase and anti-phenylethanolamine-N-methyltran- by measuring daily body weight and free cortisol in sferase Eugene Tech. Internat., Allendale, NJ, USA, morning urine [9]. At the end of the experimental period, anti-glutamate and anti-GABA Sigma, Germany; [28,34]. animals were decapitated, one subordinate and one control To co-localize immunoreactivity and receptor expression, at the same time, and brains were immediately frozen over antibody-stained sections were subjected to the in situ liquid nitrogen. hybridization procedure. H . Meyer et al. Brain Research 880 2000 147 –158 149 2.4. In situ hybridization labeled neuron was subtracted from specific labeling. Sections of a control and a stressed animal mounted on the To identify the neurons expressing a -adrenoceptor same slide were analyzed pair wise for details see Section 2A mRNA, antibody-stained paraffin sections were pretreated 3. For statistical evaluation, data were subjected to before the in situ hybridization. Briefly, slides were ANOVA and Newman–Keuls post hoc test. hydrated in graded alcohols, rinsed in 0.9 NaCl and phosphate-buffered saline PBS for 5 min each and post- fixed in 4 PFA 10 min. They were then treated with

3. Results