nNOS Expression in the Paraventricular Nucleus 335 Fig. 1. nNOS immunoreactivity and number of nNOS-ir neurons in the parvocellular and the magnocellular PVN. Rats were sacri- ficed for nNOS immunohistochemistry at 1 h after an intracere- broventricular (icv) injection of Sp-cAMP or saline vehicle. FC/veh, free-fed control rats received icv administration of 5 μl sterilized saline; FD/veh, 48 h food deprivation and received icv administra- tion of 5 μl sterilized saline; FD/Sp-cAMP, 48 h food deprivation and received icv administration of 5 μl of 40 nmol Sp-cAMP in sterilized saline; FD/Rp-cAMP, 48 h food deprivation and received icv administration of 5 μl of 40 nmol Rp-cAMP in sterilized saline; mP, medial parvocellular subdivision; pM, posterior magnocellular subdivision; PVN, paraventricular nucleus; 3v, third ventricle; *p＜ 0.05, **p＜0.01 vs. FC/veh; #p＜0.05 vs. FD/veh, Values are presen- ted as means±S.E. (Olympus Co., Tokyo, Japan) and MCID image analysis sys- tem (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 sec- tions of each rat were averaged per section, and the indivi- dual mean counts averaged across rats within experimental groups. All data were analyzed by one-way analysis of var- iance (ANOVA), and preplanned comparisons with the con- trol 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. RESULTS Food deprivation significantly reduced the number of nNOS immuno-positive (-ir) neurons in both the medial parvocellular (mP) and the posterior magnocellular (pM) PVN (Fig. 1). One hour after an icv Sp-cAMP, nNOS-ir was significantly increased in the parvocellular PVN of food de- prived rats (p＜0.05, FD/veh vs. FD/Sp-cAMP); however, this increase was not observed in the magnocellular PVN. nNOS-ir in the PVN of food deprived rats that received an icv Rp-cAMP, cAMP antagonist, did not differ from the food deprived rats that received icv saline. pCREB-ir in the parvocellular PVN of food deprived rats that received icv Sp-cAMP was increased significantly (p＜ Fig. 2. pCREB immunoreactivity and number of pCREB-ir nuclei in the parvocellular and the magnocellular PVN. Rats were food deprived for 48 h and then received icv administration of 5 μl sterilized saline (vehicle) or 40 nmol Sp-cAMP in sterilized saline, and sacrificed 1 h after the icv injections for pCREB immunohisto- chemistry. mP, medial parvocellular subdivision; pM, posterior magnocellular subdivision; PVN, paraventricular nucleus; 3v, third ventricle; *p＜0.05 vs. vehicle controls; Values are presented as means±S.E. Fig. 3. Plasma corticosterone levels at 1 h after an icv admini- stration of Sp-cAMP or saline vehicle. FC/veh, free-fed control rats received icv administration of 5 μl sterilized saline; FD/veh, 48 h food deprivation and received icv administration of 5 μl sterilized saline; FD/Sp-cAMP, 48 h food deprivation and received icv administration of 5 μl of 40 nmol Sp-cAMP in sterilized saline; *p＜0.05 vs. FC/veh, Values are presented as means±S.E. 0.05) compared with vehicle injected controls (Fig. 2). Sp- cAMP did not increase pCREB-ir in the magnocellular PVN of food deprived rats. Cardiac blood was collected at sacrifice and the plasma levels of corticosterone were analyzed by radioimmuno- assay (Fig. 3). Food deprivation significantly increased the plasma levels of corticosterone (p＜0.05, FC/veh vs. FD/veh), and Sp-cAMP administration did not alter the plasma corti- costerone levels of food deprived rats.