438 Y Kwak, et al depletion effect on the central nervous system. Grancha et al. [16] studied the role of the pontine parabrachial nucleus in conditioned taste aversion using 0.15 M NaCl. In these studies, the main concern was the difference of FLI levels in the specific NST regions before and after taste condi- tioning. From these studies, it is difficult to discriminate directly the effect of salt taste stimulation on FLI levels in the NST. There were few studies for this issue. Travers and Hu [17] reported an interesting study for the FLI ex- pression following salt taste stimulation in rats NST. The FLI levels for 0.3 M NaCl were not significantly different from control condition using distilled water (DW). The level of FLI was a little lower than control but a little higher than unstimulated condition. Meanwhile, Stratford and Finger [18] recently reported different result while they tested the taste ability of purinergic receptor in mice. The FLI levels following 0.15 M NaCl stimulation were sig- nificantly higher than DW and unstimulated condition in the overall NST. The regional difference in FLI levels along the rostral-caudal axis were also reported and salt taste showed higher FLI level. But it is not a direct comparison because they just compared FLI expressions by DW and NaCl stimulation between wild type and purinergic re- ceptor knockout mice. From these two studies, FLI levels along the rostral-caudal axis in the NST are still unclear. Therefore, the present study was conducted to verify salt concentration-dependent FLI levels in the NST. The FLI levels in the overall NST and six subregions (dorsal/ven- tral×medial/middle/lateral) of the NST were compared be- tween control (DW) and NaCl solutions with four different concentrations for salt taste stimulations. METHODS Animals All procedures in this study were conformed to guidelines set forth by the National Institute of Health and were ap- proved by the Institutional Animal Care and Use Commit- tee of Yonsei University Health System. Twenty-seven adult Sprague-Dawley rats weighting 270∼380 g were used. Rats were housed in plastic cages with a 12:12-h light-dark schedule and given free access to food and water before the taste stimulation study. All stimulation was per- formed during the light phase. Tastant stimulation Rats were anesthetized by intraperitoneal injection of urethane (1.25 g/kg, i.p.). After 30 min, rats were moved to quiet room for tastants stimulation. The rat body was turned left side up to make easy access of pipette to the tongue for tastant solution application. The tail and neck were fixed using tape to prevent body movement during taste stimulation. The tongue was gently pulled out and clamped. DW for control and NaCl (Duskan Pure Chemicals, Ansan, Korea) solutions with four different concentrations (0.1, 0.3, 0.4 and 1.0 M) was used for a tastant. The NaCl was dissolved in distilled water immediately before ex- periments. A tastant of 150μl volumes was dropped to an- terior 2/3 portion of the tongue on left side and tried to soak left and right side evenly. After 2 min of tastant appli- cation, the applied tastant was washed with DW and excess DW was absorbed using paper tissue gently after 30 sec. This tastant stimulation was performed 8 times with 4 min interval. c-Fos Immunohistochemistry After the 1 h poststimulation period, rats were injected with a lethal dose of urethane and perfused through the left ventricle with phosphate-buffered saline (PBS, pH 7.4) and then with 4% paraformaldehyde (PFA). The brain was removed from the skull, postfixed in 4% PFA for overnight. The brain block containing the NST was separated and washed three times for 5 min with PBS and stored in 10%, 20% and 30% sucrose-phosphate buffer (PB, pH 7.4) se- o quentially at 4 C overnight. The brain block was covered entirely with cryo-embed- ding media (frozen section compound FSC 22, Leica, Wetzlar, Germany) and was frozen completely in −80oC freezer and stored at −80oC until ready for sectioning. The coronal sections were cut with 8μm thickness and 32μm intervals at −20oC on a cryotome cryostat. The section was placed onto silane-coated slides glasses (5116-20F, Muto Pure Chemicals CO. LTD, Tokyo, Japan) and stored at −20oC until ready for c-Fos staining. Sections were washed three times with 0.02 M PBS and then incubated in PBS containing 0.3% hydrogen peroxide for 5 min to block endogenous peroxidase activity. Sections were then washed three times in PBS for 3 min. We fol- lowed the introduction of PK-6200 kit (Vector Laboratories, Burlingame, CA, USA) manual for other staining process. The sections were incubated with PBS-serum solution (PBS 10 ml with 2 drops serum) for 30 min and incubated with PBS containing 0.3% Triton-X, and c-Fos primary antisera (K-25, Santa Cruz Bio, Santa Cruz, CA, USA, diluted 1: 1250; 10 ml PBS＋60μl Trion-X100＋8μl c-Fos) for over- night at 4oC. The sections were then washed three times with PBS for 3 min and incubated with a biotinylated rab- bit anti-sheep secondary antibody (Vector Laboratories; 10 ml PBS＋2 drops of serum＋2 drops of biotinylate solution) for 30 min. The sections were washed three times with PBS for 3 min and incubated for 30 min with PBS containing avidin-biotinylated horseradish peroxidase complex (10 ml PBS＋2 drops of A solution＋2 drops of B solution). After three times wash with PBS for 3 min, the reaction was vi- sualized using a glucose oxidase- 3,3’-diaminobenzidine (DAB) with 5 mg DAB and 5 mg NiCl in 10 ml PBS. The reaction was terminated by washing 3 time with PBS and the reacted section on slides were dehydrated and cover- slipped. Photomicrographys were taken with a digital cam- era (Olympus DP71, Olympus, Victoria, Australia; reso- lution=1,036×1,024) with ×40 and ×100 magnification. Analysis of FLI expression in the NST Fig. 1A shows typical coronal sections of the r1 (left), i1 (middle) and i3 (right) areas along the rostral-caudal axis of the NST with ×40 magnification. The arrow in the figure just shows the direction of rostral-caudal axis. The serial sections from each animal need position calibration to com- pare their FLI levels along the rostral-caudal axis. To cali- bration the positions of section among animals, the section which showed that the NST meet the fourth ventricle (4V) was positioned as “0” and named i1 area (i: intermediate). If the position of the left and right NST in the same section had different positioning, the calibration was performed