Animals were maintained on a 12 hr light/dark cycle in the Georgia this website Health Sciences University animal care facility. Except for when specified in experiment, such as when food pellets were used as rewards, food and water were given ad libitum. All procedures relating to animal care and treatment conform to the Institutional and NIH guidelines. For behavioral tests in the study, we used male mice around

1 year old in age. These animals have been prescreened to make sure that they have normal vision and hearing capacity. Mice were perfused transcardially with 4% paraformaldehyde (PFA) in 1× PBS followed by a postfixation in 4% PFA overnight. Coronal sections (50 μm thick) were cut on a vibratome and collected in 0.5% PFA in 1× PBS and stored at 4°C before use. For double-immunofluorescent staining of β-galactosidase and TH, sections were incubated at 4°C overnight with gentle shaking in primary Selleck 3-Methyladenine antibody (anti-β-galactosidase [pAb] 1/5,000, Invitrogen; anti-TH [monoclonal antibody] 1/1,000) in a buffer containing 0.05% Tween 20, 10% normal goat serum, and 1× PBS following preincubation in 10% normal goat serum and 1× PBS at room temperature for 2 hr. The sections were then incubated with Alexa-conjugated secondary antibodies (1/200; Invitrogen) at room temperature for 2 hr. β-Galactosidase IR was visualized by Alexa 568 and TH IR by Alexa 488. A similar

procedure was employed to double stain NMDAR1 and TH except that anti-NR1 (polyclonal antibody 1:100; Chemicon, Temecula, CA, USA) was used as the primary antibody for NMDAR1. The sections were incubated with ever Alexa-conjugated secondary antibodies (1/200; Invitrogen) at room temperature for 2 hr. NMDAR1 was visualized by Alexa 488 and TH IR by Alexa 594. Fluorescent images were captured with a confocal

laser-scanning microscope and an epifluorescence microscope. This apparatus consists of a center platform (5 × 5 cm) 37 cm off the ground with four branching arms (30 cm long and 5 cm wide). Two of the four arms are open, and the other two arms are enclosed by black walls (20 cm high). Testing was performed during light phase in a dimly lit room (50 lux). Animals were placed on the center platform and scored for arm entries and time spent in each arm. Percentages of time animals spent in the open arms were calculated as the final readout of anxiety. Unpaired t tests were used to compare the significance between the different genotypes. RotaRod analysis was performed using the mouse version of ROTA-ROD manufactured by San Diego Instruments (San Diego, CA, USA). Mice were trained by allowing them to run on a rotarod rotating at 30 rpm for a total time span of 5 min. (Time counting was stopped when mice dropped until they were put back onto the rotarod again.) During the tests mice were again placed on top of the rotarod, which rotated at 30 rpm. Durations of each mouse that stayed on the rotarod (latency to fall) were recorded.