Results Directory UMM :Data Elmu:jurnal:B:Brain Research:Vol885.Issue1.2000:

72 T 30, wt vol and sectioned as a 1 in 12 series at 30 mm 2.4. Data analysis on a sliding microtome Fisher Scientific, Springfield, N.J. 07081 while frozen with crushed dry ice. Sections were The average number of neurons counted in each brain- stored in cryoprotectant [35] solution at 2208C to preserve stem CAergic area e.g., A1 C1, A2 C2 in studies of the immunogenicity until the time of staining. percent of brainstem neurons retrogradely labeled with Double label immunocytochemical staining for Fos and FAu after FAu injection to the PVN was: A1 C1 — 307, either CRH or tyrosine hydroxylase TH, FAu and CRH A2 C2 — 275, A5 — 231 and A6 — 1615. The average or TH, and GR and TH was accomplished using methods number of neurons counted in studies of the correlation of previously reported [16]. Once the staining procedures Fos activation of PVN CRH-immunoreactive neurons vs. were well established on practice tissues, one well of Fos activation of brainstem CAergic area neurons over a sections i.e., 1 12th of all sections cut of brainstem range of hypoxemia were PVN CRH — 347, A1 C1 — and or of hypothalamus from each animal were immuno- 651, A2 C2 — 597 and A6 — 539. stained for each pair of antigens listed in the previous Counting of coincidence of Fos and TH in brainstem sentence. Staining of the first antigen in double reactions A1 C1, A2 C2 and locus coeruleus A6 neuronal areas was accomplished with the avidin–biotin complex ABC and coincidence of Fos and CRH in PVN was done under method Elite Kits, Vector Labs, Burlingame, CA 94010 light microscopy by an observer unaware of treatment. and immunoperoxidase detection with nickel–diaminoben- Counts were expressed as a single independent value i.e., zidine Ni–DAB as the chromogen. The presence of percentage of total CA or CRH neurons expressing Fos per antigen was detected as a blue black product, which could brainstem neuronal area e.g., A1, A2 or PVN, per animal. be eliminated by substitution of normal rabbit serum for The percentages of Fos and TH coincidence for each area the primary antibody or preabsorption of the primary of CAergic brainstem neurons vs. the percent of Fos and antibody with 10 mg of the antigen per ml of antibody. CRH coincidence in neurons of the PVN were plotted Fig. Incubations with primary antisera: CRH polyclonal, gift 7 and subjected to the Pearson Product Moment Correla- of Dr A. J. Silverman 1:40,000; FAu Chemicon, poly- tion test with P-value set at 0.05. All data are expressed as clonal 1:100,000; TH Chemicon, monoclonal 1:250,000; mean6S.E.M. Fos Oncogene Sciences, AB-2 or Dr T. Curran’s alu-Fos All procedures were approved by the Cornell University antibody 1:50,000 and GR gift of Dr D. DeFranco, Animal Care and Use Committee. All facilities were BUGR2 1:70,000 were performed for 48 h at 48C. After approved by the American Association for the Accredita- development of the first antibody the tissue was rinsed and tion of Laboratory Animal Care. the second primary antiserum was applied. Afterward the tissue was again processed for ABC immunoperoxidase staining, but the chromogen used was DAB alone. The results obtained then had either black nuclei GR or Fos

3. Results

or black cytoplasmic granules FAu with brown cyto- plasm CRH or TH. Sections of adult rat hypothalamus At 40 and 60 min of the hypoxemic challenge, maternal served as positive controls. All sections to be compared carotid arterial blood values for PO in the HYPOX group 2 within an experiment were processed simultaneously. ewes which were catheterized n56 ranged from 24.4– In a subset of tissues, we performed triple labeling to 55.4 and 25.3–39.2 Torr, respectively. We know from assess the presence of GR in CA neurons that also were previous experiments T.J. McDonald, unpublished data retrogradely labeled with FAu. In those instances, the and from the extensive characterization of the production approach used by Rinaman et al. [30] was employed. of fetal hypoxemia via maternal tracheal gas infusion [12] Briefly that strategy consisted of first staining for GR with that decreasing maternal PO via infusion of N to the 2 2 ABC peroxidase and NiDAB visualization, then the sec- maternal tracheal catheter to approximately 40 Torr yields tions were reacted for the presence of TH using a final a corresponding PO decrease in our fetuses of 10–14 2 concentration of the mouse monoclonal antibody of Torr. 1:100,000. After incubation we then applied an alkaline All the TH-immunopositive neurons of the A1 C1, A2 phosphatase-conjugated anti-mouse secondary antibody. C2, A5 n. olivaris superior and A6 catecholaminergic The alkaline phosphatase was then reacted with Vector Red brainstem areas demonstrated some degree of GR immuno- Vector Laboratories according to package inserts. The reactivity, but while all GR immunoreactivity was found to tissue was then rinsed and reacted with the anti-FAu be nuclear, as opposed to cytoplasmic, not all nuclei were antiserum. After incubation with that antibody, biotin entirely filled with GR Fig. 1. enhancement according to Berghorn et al. [3] was used Brains collected after injection of FAu into the PVN with streptavidin–Bodipy Molecular probes as the fluoro- Figs. 2, 3 exhibited retrograde FAu immunostaining in phore reagent to reveal the FAu. In so doing, the GR cytoplasmic granules in many of the CA i.e., TH-im- appeared as black in the nuclei, the TH was faintly pink munopositive neurons of the brainstem. Percentages of under brightfield optics and was brightly red fluorescent, retrogradely labeled CA-immunopositive neurons from and the FAu fluoresced green. FAu injected fetuses n53 are summarized in Fig. 4; as T .J. McDonald et al. Brain Research 885 2000 70 –78 73 Fig. 1. Micrographs of the various brainstem CA cell areas double labeled for GR black nuclei and TH brown cytoplasm. In A1, A2, A5 and A6, all the TH-immunopositive neurons showed staining for GR, but not all nuclei were entirely filled with GR arrows. Scale bar5100 mm. depicted the largest proportion were found in A1 C1 was significantly correlated with Fos activation in A1 C1 2 2 followed by A6, A5 and A2 C2 CAergic areas. r 50.894; P,0.005 and A2 C2 r 50.848; P,0.002, 2 In order for CAergic neurons of the brainstem to be in a but not A6 C6 r 50.636; P50.065 areas. position to influence the physiology of PVN neurons it was important to establish that neurons of this phenotype also contain GR and project fibers to the PVN. Therefore triple

4. Discussion