334 SB Yoo, et al ing-induced elevation of the plasma glucocorticoids is re- stored shortly after refeeding [5,15]. These reports together suggest that refeeding-induced nNOS expression and CREB phosphorylation in the PVN may require glucocorticoid dis-inhibition. However, our previous study demonstrated that glucocorticoid dis-inhibition alone is not sufficient to induce nNOS expression in the PVN [5]. It was reported that food deprivation decreases the hypothalamic cAMP levels and refeeding normalizes it [16]. In this study, we demonstrate that the intracerebroventricular (icv) injection of the membrane-permeable cAMP-dependent protein kinase A (PKA) activator Sp-cAMP (adenosine-3’,5’-cyclic mono- phosphorothioate Sp-isomer) without food supplement res- cues the fasting-induced decrease of nNOS expression in the PVN without affecting the plasma corticosterone levels. METHODS Animals 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±1oC), 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 pu- rified 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 experi- ments were approved by the Committee for the Care and Use of Laboratory Animals at Seoul National University. Intracerebroventricular (icv) cannulation 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 ven- tricle (1.2 mm caudal to bregma, 1.5 mm lateral to the mid- line, 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 in- serted into each guide cannula and remained in place ex- cept 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 recov- ery, 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 re- sponded 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. Drug treatments 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/ve- hicle, FD/Sp-cAMP or FD/Rp-cAMP groups underwent a 48 h of food deprivation beginning at 09:00 h, and then re- ceived 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 steri- lized 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). Immunohistochemistry One hour after the drug injections, rats were anesthetized with over doses of sodium pentobarbital (Hallym Pharma- ceutical 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, slid- ing 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 (Vectas- tain 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 an- ti-pCREB, which recognizes p43 phosphorylated CREB (1： 1,000 dilution, Upstate biotechnology, NY, USA), antibodies were used as primary antibodies, and biotinylated anti-rab- bit 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. Plasma corticosterone assay 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 －80oC until used for the assay. Plasma cortico- sterone 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]. Statistical analysis The number of nNOS immunopositive cells was blind- counted by hand, pCREB immunopositive nuclei auto- counted, 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