Materials and methods staining with anti-Fos and anti-vasopressin antibodies; this

96 Y inhibited by intravenous injection of vasopressin antago- Cruz Biotechnology, Santa Cruz, CA, USA in PBS nist, suggesting that senktide-induced pressor response was containing 1 normal donkey serum NDS, 0.3 Triton mediated by release of vasopressin [12,28]. Moreover, X-100 and 0.03 NaN overnight at room temperature. 3 immunohistochemical and in situ hybridization studies The sections were then washed in PBS and incubated with have revealed that NK3 receptor is widely distributed in biotinylated goat anti-rabbit IgG 1:200; Vector Laborator- the central nervous system of the rat especially in the ies, Burlingame, CA, USA in the NDS incubation medium hypothalamus [9,15], and that a large proportion of NK3 for 3–4 h, washed again, and reacted with an Elite ABC receptor-expressing neurons in the paraventricular and Kit 1:200; Vector in PBS for 2–3 h. The sections were supraoptic nuclei of the hypothalamus contain vasopressin finally reacted with 0.02 diaminobenzidine and 0.003 [10]. However, the role of NK3 receptor in the modulation hydrogen peroxide in 0.05 M Tris–HCl buffer pH 7.6 of release of vasopressin from the hypothalamus is not with nickel intensification. fully understood. Therefore, in the present study, experi- ments were designed to examine the effects of intracere- 2.3. Double labeling for Fos and vasopressin broventricular administration of senktide on Fos expression in immunohistologically identified vasopressin-containing Since injection of 0.5 mg of senktide-induced maximal neurons in the rat hypothalamus. Fos expression in the hypothalamus see Section 3, one series of sections from the rats with injection of 0.5 mg senktide was subjected to double immunohistochemical

2. Materials and methods staining with anti-Fos and anti-vasopressin antibodies; this

made possible the evaluation of the number of vasopressin- 2.1. Preparation of animals and tissue containing neurons activated by the treatment. The sections were first processed for Fos immunohistochemistry by the Twenty-two male rats Wistar weighing 200–250 g PAP method: they were incubated with rabbit anti-Fos were used in the present study. They were housed in a antibody 1:2000; Santa Cruz Biotechnology in the NDS temperature-controlled room 22628C on a 12:12-h incubation medium overnight and then with goat anti- 07:00–19:00 h light–dark cycle, with food and water rabbit IgG 1:100; Vector in the same medium for 6 h. freely available. All protocols used in the present study Finally, the sections were reacted with rabbit PAP-complex have been approved by the Committee of Animal Use for 1:100; Sigma for 4 h. The bound peroxidase was Research and Education of the Fourth Military Medical visualized by incubation with 0.02 DAB and 0.003 University, China. The rats were anesthetized by intra- H O for 20–30 min with nickel intensification. After 2 2 peritoneal injection of sodium pentobarbital 40 mg kg several washes in PBS, the sections were immunostained body weight and fitted by stereotaxic surgery with a for vasopressin by the ABC method. The sections were stainless-steel guide cannula aimed at the lateral ventricle incubated with mouse anti-vasopressin antibody 1:100; [26]. Five to 6 days after the operation, the rats were PS-41; Gift from Dr. H. Gainer [1,38] in the NDS anesthetized in the same way and senktide Sigma, St. incubation medium overnight. The specificity of this Louis, MO, USA dissolved in 8 ml saline was injected antibody has been verified by various methods, and this into the lateral ventricle through a stainless-steel injector antibody could distinguish neurons containing vasopressin temporarily inserted into the guide cannula. The doses of from those with oxytocin [1,38]. After being washed in senktide were 0, 5 ng, 50 ng, 0.5 mg and 5 mg n54 for 0, PBS, the sections were incubated with biotinylated donkey 5 ng, 50 ng and 5 mg, respectively; n56 for 0.5 mg. anti-mouse IgG 10 mg ml; Jackson Immunochemicals, Ninety minutes later, the rats were perfused with 100 ml of West Grove, PA, USA for 8 h at room temperature, and 0.01 M phosphate-buffered saline PBS; pH 7.4, followed then were treated with the Elite ABC Kit 1:200; Vector by 500 ml of 4 paraformaldehyde and 0.1 picric acid in for 4 h at room temperature. To discriminate vasopressin- 0.1 M phosphate buffer PB; pH 7.4. All the perfusions in like immunoreactivity -LI from Fos-LI, the bound per- the present study were carried out between 11:00 and oxidase for vasopressin-LI was visualized with 0.02 12:00 h on the day the experiment was performed. The DAB and 0.003 hydrogen peroxide in Tris–HCl buffer brains were removed immediately and placed in 20 pH 7.6 without nickel intensification. In the control sucrose in PB overnight at 48C. The brains were cut into experiments, when one of the primary antibodies was five series of 30-mm thick transverse sections on a freezing omitted or replaced with normal rabbit for Fos immuno- microtome. histochemistry or mouse for vasopressin immunohisto- chemistry IgG, no immunoreactivity was detected in the 2.2. Fos immunohistochemistry control sections. Fos was identified using the avidin–biotin immuno- 2.4. Quantitative analysis histochemical protocol. Briefly, one series of sections were incubated with rabbit anti-Fos antibody 1:4000; Santa The number of neurons containing Fos-LI was counted Y .-Q. Ding et al. Brain Research 882 2000 95 –102 97 from the microscope in selected nuclei unilaterally in one 3. Results series of sections 12–14 sections; see Section 2.2 from each rat; these serial sections covered whole extent of the 3.1. Fos expression in the hypothalamus paraventricular and supraoptic nuclei of the hypothalamus. Data were expressed as mean6S.E.M. In the double In rats injected with 8 ml saline as control, Fos-like immunostaining experiment, the number of cells labeled immunoreactive neurons were present consistently in very for vasopressin, Fos, and double-labeled cells were low numbers in several ares of the hypothalamus. These counted in one series of sections 12–14 sections; see areas included the lateral preoptic area, paraventricular Section 2.3 in each of six rats, and the percentages of hypothalamic nucleus, and supraoptic nucleus Fig. 3. double-labeled neurons from vasopressin- and Fos-positive In contrast, injection of senktide into the lateral ventricle neurons in the hypothalamus paraventricular nucleus, resulted in an obvious increase of Fos expression in the supraoptic nucleus, circular nucleus, and lateral hypo- hypothalamus Figs. 1–3. Fos-positive neurons were thalamic perivascular nucleus were calculated for each rat, present with a high density in the medial and lateral and then averaged. magnocellular parts of the paraventricular nucleus and the Fig. 1. Drawings of transverse sections through the hypothalamus, showing the distributions of Fos filled circles, vasopressin asterisks, and Fos vasopressin double-labeled neurons crosses. Rat was sacrificed 90 min after injection of 5 mg of senktide into the lateral ventricle. Each symbol in the circular nucleus NC and lateral hypothalamic perivascular nucleus LHP represents one neuron, but in the other regions one symbol presents three neurons. 3V, third ventricle; ac, anterior commissure; apPV, anterior parvicellular part of the paraventricular nucleus; Ar, arcuate nucleus; DM, dorsomedial hypothalamic nucleus; f, fornix; ic, internal capsule; lpPV, lateral parvicellar part of the paraventricular nucleus; mmPV, anterior magnocellular part of the paraventricular nucleus; mpPV, medial parvicellular part of the paraventricular nucleus; op, optic chiasm; pmPV, posterior magnocellular part of the paraventricular nucleus; SOR, retrochiasmatic part of the supraoptic nucleus; SCh, suprachiasmatic nucleus; SO, principal part of the supraoptic nucleus; VM, ventromedial hypothalamic nucleus; ZI, zona incerta. 98 Y Fig. 2. Photomicrographs showing distribution of Fos-positive neurons in the paraventricular nucleus a, principal part of the supraoptic nucleus SO b, retrochiasmatic supraoptic nucleus SOR c, lateral hypothalamic perivascular nucleus d, circular nucleus NC e, and arcuate nucleus Ar f of the hypothalamus. Rat was sacrificed 90 min after intracerebroventricular injection of 0.5 mg of senktide. Asterisks indicate a relatively large blood vessel in the lateral anterior hypothalamus. mpPV, medial parvicellular part of the paraventricular nucleus. Bar560 mm. principal part of the supraoptic nucleus. With a lower In addition, many cells in the ependyma of the lateral density they were seen in the retrochiasmatic part of the and third ventricles and choroid plexus showed Fos-LI supraoptic nucleus, arcuate nucleus and circular nucleus. A after the intraventricular injections of senktide but not after few Fos-positive neurons were seen in the lateral preoptic injection of saline Fig. 2a. area, suprachiasmatic nucleus, anterior hypothalamic area, anterior magnocellular part of the paraventricular nucleus, parvicellular part of the paraventricular nucleus, periforni- 3.2. Double labeling cal region, posterior hypothalamic area, and along rela- tively large blood vessels lateral hypothalamic perivascu- The distribution of vasopressin-like immunoreactivity lar nucleus [11] in the anterior hypothalamic area Figs. 1 was as previously reported [29,33–35]. In double immuno- and 2. Few Fos-positive neurons, if any, were seen in the stained sections, neurons with both Fos-LI and vasopres- dorsomedial and ventromedial nuclei of the hypothalamus. sin-LI were found in the hypothalamus. Double-labeled In addition, administration of 5 ng of senktide increased neurons were unequivocally distinguishable from single- Fos expression in the hypothalamus, but the number of labeled ones by the Fos black reaction product in cell Fos-positive neurons reached maximum at a dose of 0.5 nuclei and the homogeneous brown color vasopressin stain mg; a higher dose of senktide 50 mg failed to increase in the cytoplasm Fig. 4. Double-labeled neurons were Fos expression further Fig. 3. Moreover, the distribution primarily found in the medial and lateral magnocellular patterns of Fos-positive neurons in the hypothalamus parts of the paraventricular nucleus, principal part of the among the rats treated with different doses of senktide supraoptic nucleus, and to a lesser degree in the parvicellu- were almost the same. lar part of the paraventricular nucleus, retrochiasmatic part Y .-Q. Ding et al. Brain Research 882 2000 95 –102 99 nucleus and lateral hypothalamic perivascular nucleus were 76, 84 and 90, respectively Fig. 5.

4. Discussion