Sprague-Dawley rats were purchased (Samtako Bio, Osan, Korea), and cared in a specific-pathogen-free (SPF) barrier area with constant control of temperature (22±1℃), humidity (55%), and a 12/12 hr light/dark cycle (lights-on at 07:00 AM). Standard laboratory food (Purina Rodent Chow, Purina Co., Seoul, Korea) and membrane filtered purified water were available ad libitum. Animals were cared according to the Guideline for Animal Experiments, 2000, edited by the Korean Academy of Medical Sciences, which is consistent with the NIH Guidelines for the Care and Use of Laboratory Animals, revised 1996. All animal experiments were approved by the Committee for the Care and Use of Laboratory Animals at Seoul National University.

Under chloral hydrate (153 mg/kg) and pentobarbital (35 mg/kg) anesthesia, rats (280~300 g) were stereotaxically implanted with a 22-gauge, stainless steel guide cannula (Plastics One, Roanoke, VA) aimed towards the lateral ventricle (1.2 mm caudal to bregma, 1.5 mm lateral to the midline, and 4 mm below the skull surface). Guide cannulae were held in place with dental acrylic bonded to stainless steel screws anchored to the skull. An obdurator was inserted into each guide cannula and remained in place except during injections when it was removed and replaced with an injector that extended 1.0 mm beyond the tip of the guide cannula. After 1 week of post-operational recovery, patency and placement of the cannula was verified by injection of 100 ng human angiotensin II (Sigma Chemical Co., St Louis, MO, USA) dissolved in 5 µl of 0.15 M NaCl; rats with cannulas projecting into the lateral ventricle responded to the angiotensin injection by vigorously licking the water bottle within 2 min [17-19], while rats that failed to drink were dropped from the study. Cannula placements were also verified postmortem by sectioning through the brain.

Rats verified for the cannula placement were divided into four different groups (n=6 in each group, total 24 rats); i.e, FC/vehicle (saline icv with ad libitum access to food), FD/vehicle (saline icv following food deprivation), FD/Sp-cAMP (Sp-cAMP icv following food deprivation) and FD/Rp-cAMP (Rp-cAMP icv following food deprivation). Rats in FD/vehicle, FD/Sp-cAMP or FD/Rp-cAMP groups underwent a 48 h of food deprivation beginning at 09:00 h, and then received an icv injection of Sp-cAMP (Sp-8-bromo-cAMP, Sigma Chemical Co., St Louis, MO, USA; 5 µl of 40 nmol in sterilized saline), Rp-cAMP (Rp-8-bromo-cAMP, Sigma Chemical Co., St Louis, MO, USA; 5 µl of 40 nmol in sterilized saline) or sterilized saline (5 µl of 0.15 M NaCl). Sp-cAMP, Rp-cAMP or saline was delivered through the icv guide cannulas over 30 s with a handheld 50 µl syringe (Hamilton Co., Reno, NV, USA). The injector was left in place for 30 s after solution delivery. Free-fed control group (FC/vehicle) received an icv injection of sterilized saline (5 µl of 0.15 M NaCl).

One hour after the drug injections, rats were anesthetized with over doses of sodium pentobarbital (Hallym Pharmaceutical Co., Seoul, Korea), and transcardiac perfusions were performed first with heparinized isotonic saline then with ice-cold 4% paraformaldehyde (Sigma Co., MO, USA) in 0.1 M sodium phosphate buffer. Brains were immediately dissected out, blocked, post-fixed for 2 h, and transferred into 30% sucrose (Sigma Co., MO, USA) for cryoprotection. Forty-micron coronal sections were cut on a freezing, sliding microtome (HM440E, Microm Co., Germany). Alternate sections were collected throughout the rostrocaudal extent of the hypothalamic PVN (between bregma - 1.3 mm and - 2.1 mm) [20]. Immunohistochemistry was performed with standard DAB reaction using commercial ABC kit (Vectastain Elite Kit, Vector Laboratories, CA, USA) as previously described [21]. Polyclonal rabbit anti-nNOS, which reacts with the 160 kDa nNOS protein and does not exhibit any cross-reactivity with the related eNOS or iNOS proteins (1:2,000 dilution, Zymed Laboratories, CA, USA), and anti-pCREB, which recognizes p43 phosphorylated CREB (1:1,000 dilution, Upstate biotechnology, NY, USA), antibodies were used as primary antibodies, and biotinylated anti-rabbit IgG (1:200 dilution, Vector Laboratories, CA, USA) as secondary. Immuno-stained sections were mounted in an anatomical order onto gelatin-coated slides from 0.05 M phosphate buffer, air-dried, dehydrated through a graded ethanol to xylene, and cover-slipped.

Cardiac blood was rapidly collected in the heparinized tube immediately after exposing the heart for perfusion, and centrifuged at 2,000 rpm for 20 min. The plasma was transferred into new tubes, frozen in liquid nitrogen, and stored at -80℃ until used for the assay. Plasma corticosterone levels were determined by radioimmunoassay using ¹²⁵I-labelled Coat-A-Count kit (DPC, CA, USA). All rats were sacrificed between 10:00 h and 12:00 h to minimize diurnal variation in the plasma levels of corticosterone [22].

The number of nNOS immunopositive cells was blind-counted by hand, pCREB immunopositive nuclei autocounted, after digitizing 720×540 micron images of three sections from the PVN (closest sections to bregma - 1.88 mm) from each brain using an Olympus BX-50 microscope (Olympus Co., Tokyo, Japan) and MCID image analysis system (M2, Imaging Research Inc., Ontario, Canada). nNOS manual count was done blind by S.B.Y. and confirmed by a separate blind count by J.Y.L. Cell counts from the sections of each rat were averaged per section, and the individual mean counts averaged across rats within experimental groups. All data were analyzed by one-way analysis of variance (ANOVA), and preplanned comparisons with the control performed by post-hoc Fisher's PLSD and presented by means±SE. For all comparisons, the level of significance was set at p≤0.05.