Alanced across the left and right sides of the testing cages. Water and sucrose intakes were measured during the 12-h dark period by weighing bottles before and after the test. Tests were performed on 2 consecutive days. Sucrose preference was calculated as the percentage of sucrose consumed. (3) Passive avoidance test (PAT). After 7 days of acclimation to cage and diet, all mice were subjected to PAT to examine the basal performance of learning and Title Loaded From File memory prior to CUS exposure. The apparatus for this test consisted of two compartments, one light and the other dark, separated by a vertical sliding door [21]. A mouse was initially placed in the light compartment for 30 sec. Then, the door was opened to permit the mouse to enter the dark compartment. After the mouse entered the dark compartment, the door was closed. Thirty seconds later, the mouse was given a 0.2 mA electric shock for 2 sec. The mouse was allowed to recover for 30 sec and then returned to the home cage. Twenty-four hours later, the mouse was again placed in the light compartment and the door was opened. The latency time until the mouse stepped through the door was determined as an index of learning and memory. To examine time-course changes in learning and memory, the latency to enter the dark compartment was measured every week during the CUS procedure. (4) Object recognition test (ORT). ORT was used to examine recognition memory [22,23]. After the mice were transferred to a cage for the ORT and acclimated for 24 h, they were exposed to two differently shaped objects for 10 min. The number of actions of exploring and/or sniffing two objects was counted for the initial 5-min period (Training). The next day, to examine memory Title Loaded From File Retention, one of the original objects was replaced with a novel one with a different shape, and then the number of actions of exploring and/or sniffing the novel object was counted for 5 min (Retention). The recognition index was calculated by dividing the number of actions of exploring and/or sniffing the novel object by the total number of actions of exploring and/or sniffing (novel object+familiar object) [23].vibratome (VT 1000S, Leica Microsystems, Germany) at 40 mm. Serial sections were immersed in PBS. Ninety-six-well plates were used to maintain the correct order of the sections in PBS at 4uC. The right hemisphere was divided into the hippocampus, cerebral cortex, hypothalamus and cerebellum. These samples were quickly frozen in liquid nitrogen and stored at 280uC until analysis. (2) BrdU and Ki67. BrdU- or Ki67-positive cells were identified immunohistochemically. The sections were incubated with 3 hydrogen peroxide in methanol to block endogenous peroxidase activity. BrdU sections were incubated with 2 M HCl for 30 min at 37uC and M.O.M. mouse IgG blocking solution for 1 h. Ki67 sections were exposed to heat (100uC) in 100 mM citric acid buffer (pH 6.0) for 5 min using a microwave for antigen retrieval and the sections were then blocked with normal goat serum. After washing with PBS, the sections were incubated for two nights with the primary antibody, a mouse monoclonal antiBruU antibody (BD Pharmingen, 1:200) or rabbit polyclonal antiKi67 antibody (Abcam, 1:500). After washing, the BrdU or Ki67 sections were incubated with anti-mouse biotinylated IgG secondary antibody (Vector Laboratories, 1:250) or goat antirabbit biotinylated IgG (Vector Laboratories, 1:100) for 2 h at room temperature, respectively. Both BrdU and Ki67 sections were i.Alanced across the left and right sides of the testing cages. Water and sucrose intakes were measured during the 12-h dark period by weighing bottles before and after the test. Tests were performed on 2 consecutive days. Sucrose preference was calculated as the percentage of sucrose consumed. (3) Passive avoidance test (PAT). After 7 days of acclimation to cage and diet, all mice were subjected to PAT to examine the basal performance of learning and memory prior to CUS exposure. The apparatus for this test consisted of two compartments, one light and the other dark, separated by a vertical sliding door [21]. A mouse was initially placed in the light compartment for 30 sec. Then, the door was opened to permit the mouse to enter the dark compartment. After the mouse entered the dark compartment, the door was closed. Thirty seconds later, the mouse was given a 0.2 mA electric shock for 2 sec. The mouse was allowed to recover for 30 sec and then returned to the home cage. Twenty-four hours later, the mouse was again placed in the light compartment and the door was opened. The latency time until the mouse stepped through the door was determined as an index of learning and memory. To examine time-course changes in learning and memory, the latency to enter the dark compartment was measured every week during the CUS procedure. (4) Object recognition test (ORT). ORT was used to examine recognition memory [22,23]. After the mice were transferred to a cage for the ORT and acclimated for 24 h, they were exposed to two differently shaped objects for 10 min. The number of actions of exploring and/or sniffing two objects was counted for the initial 5-min period (Training). The next day, to examine memory retention, one of the original objects was replaced with a novel one with a different shape, and then the number of actions of exploring and/or sniffing the novel object was counted for 5 min (Retention). The recognition index was calculated by dividing the number of actions of exploring and/or sniffing the novel object by the total number of actions of exploring and/or sniffing (novel object+familiar object) [23].vibratome (VT 1000S, Leica Microsystems, Germany) at 40 mm. Serial sections were immersed in PBS. Ninety-six-well plates were used to maintain the correct order of the sections in PBS at 4uC. The right hemisphere was divided into the hippocampus, cerebral cortex, hypothalamus and cerebellum. These samples were quickly frozen in liquid nitrogen and stored at 280uC until analysis. (2) BrdU and Ki67. BrdU- or Ki67-positive cells were identified immunohistochemically. The sections were incubated with 3 hydrogen peroxide in methanol to block endogenous peroxidase activity. BrdU sections were incubated with 2 M HCl for 30 min at 37uC and M.O.M. mouse IgG blocking solution for 1 h. Ki67 sections were exposed to heat (100uC) in 100 mM citric acid buffer (pH 6.0) for 5 min using a microwave for antigen retrieval and the sections were then blocked with normal goat serum. After washing with PBS, the sections were incubated for two nights with the primary antibody, a mouse monoclonal antiBruU antibody (BD Pharmingen, 1:200) or rabbit polyclonal antiKi67 antibody (Abcam, 1:500). After washing, the BrdU or Ki67 sections were incubated with anti-mouse biotinylated IgG secondary antibody (Vector Laboratories, 1:250) or goat antirabbit biotinylated IgG (Vector Laboratories, 1:100) for 2 h at room temperature, respectively. Both BrdU and Ki67 sections were i.
