and memory remains unclear. Here we provide evidence that TLR4 is involved in modulating hippocampus-dependent learning and memory. By measuring performance of TLR4-deficient (TLR42/2) and wild-type (TLR4+/+) mice in tests of hippocam- pus-dependent cognitive function, we provide evidence that developmental TLR4 deficiency enhances spatial reference memory but impairs contextual fear conditioning. In contrast, antagonism of TLR4 in adult TLR4+/+ mice has no impact on cognition, and instead affects anxiety responses. Methods Animals Young adult male congenic TLR42/2 mice (B6.B10ScN- Tlr4lps–del/JthJ) (n = 19) and their respective wildtype mice (C57bl/6) (n = 24) were purchased from Jackson Laboratories (Bar Harbor, ME, USA). The TLR42/2 mice we used were backcrossed to C57bl/6 mice for 6 generations in the original laboratory where they were generated, as well as backcrossed an additional 7th generation in the Jackson Laboratory, followed by 7 inbreeding generations (see: http://jaxmice.jax.org/strain/ 007227.html). All experiments were completed using mice at 2– 4 months of age. Animal care and experimental procedures followed NIH guidelines and were approved by the National Institute on Aging Animal Care and Use Committee. Behavioral testing Spatial learning. To evaluate spatial learning and memory, mice were tested in variants of the Morris Water Maze (MWM) paradigm [10]. In all spatial learning tests, mice were first tested for their performance in a visible platform variant to exclude the possibility that the tested mice are impaired in visual ability or motivation, and to facilitate their habituation to the water pool. Mice were trained in the a water-filled pool (160 cm diameter) for 3 consecutive days, 4 trials per day, without spatial cues on the walls of the room, but with a black metal object attached to a visible platform (153 cm2 area), which was located above water- level. Mice were allowed to swim freely for 60 s, and mice unable to find the visible platform were placed on the visible platform for 10 seconds. To test for spatial reference memory, mice were trained in the pool for 5 consecutive days, 4 trials per day, with spatial cues on the walls of the room. The cues (see [10] for details) were black and white only, to reduce possible effects of color discrimination capabilities between the different genotypes. The platform was hidden 0.5 cm below the surface of the water; the water was made opaque using nontoxic white paint to prevent the mice from seeing the platform. The water was maintained at a temperature of 2760.5uC, and constant temperature was ensured by mixing the water every 1.5 hours, and supplementing warm water when needed. The platform location was constant and starting points were changed every trial to avoid track memorization. When trials ended, either when the mouse had found the platform or when 60 seconds passed, mice were allowed to rest on the platform for 30 seconds. After completion of the 5- day learning phase, mice were tested for memory retention of the platform location. The platform was removed and mice were allowed to swim freely for 60 seconds. These ‘probe trials’ were conducted 24 and 48 hours after the last trial and every day afterwards until memory of the platform location was extinct in all experimental groups, which was indicated by equal mean distance from the platform when compared between groups. Data on mean distance from the platform as well as all other criteria was provided by the Anymaze software. TLR4 in Learning In the visible platform, reference and working memory variants of the MWM, tests were conducted under conditions of 20 lux to reduce stress to the mice. In experiment involving cannulations, the cannulated mice were allowed 7 days to recover from the surgery before testing in the MWM. In all the MWM tests, mice that exhibited passivity or thigmotaxic swimming pattern were excluded from analysis [10]. Open Field Test. Open field arena testing was performed using the MEDOFA-MS system (Med Associates, St Albans, VT, USA). Animals were placed in the center of an open field (40.64 cm 640.64 cm) and exploration was assessed for 15 min. Cages were cleaned with ethanol following each session. The peripheral 10.16 cm of the zone were considered as the peripheral zone and the central 20.32 cm2 were considered as the central zone. All open field tests were conducted under light intensity of 400 1ux. Rotarod test. Rotarod tests were performed using the ENV- 577M system (Med-associates, St. Albans, VT, USA), in a brightly lit room (1300 lux). Rotarod acceleration was set to 4–40 revolutions per minute (RPM). Mice were placed on the rotarod for three 5-minute trials with 15 minutes rest between trials, and time until the first fall was recorded. The apparatus was routinely cleaned with water and ethanol following each session. Fear Conditioning. Before testing, mice were first habituated to the testing room for 3 h/day for 3 days. In the training session, mice were placed in a contextual conditioning chamber (model MEDVFC-NIR-M; Med Associates) placed inside sound attenu- ating boxes (model MED). The conditioning chambers contained a metal grid on the floor (context A) and mice were allowed to explore the chamber for 2 min. At the end of 2 min mice were subjected to three sessions of audio tone (CS, conditioned stimulus) and foot shock (US, unconditioned stimulus). Audio tone (5 kHz, 70 dB) was on for 30 s, followed immediately by a 0.5-mA, 2-s foot shock from the metal grid floor. Thirty seconds separated each session. Foot shock intensity was determined in a preliminary test on a separate cohort of animals for the minimal applicable intensity to achieve a minimal freezing threshold of 40% during contextual fear. On the following day, in the contextual fear session (context A), mice were returned to the conditioning chamber for 5 min without any shock or audio tone. The percentage of time freezing was recorded and used as an index of contextual memory. In the cued conditioning (conducted 3 h following contextual conditioning) mice were returned to the chamber but in a different context (context B). Context B was comprised of plastic triangle inserted to the cage coupled with plastic flooring placed underneath the triangular plastic insert. Mice were allowed to explore the chamber for 5 min without any audio tone. Following this, five audio tones were played for 30 s each. The percentage of time freezing until and after the audio tones was recorded and used as an index of cued memory. In all fear conditioning tests, cages were cleaned with ethanol between tests. Elevated Plus Maze. The apparatus consisted of a plus-sign shaped maze elevated 60 cm from the floor and comprised of two opposite open arms, 25 cm 65 cm each, crossed by two arms of the same dimensions enclosed by 30-cm-high walls. In addition, a 1-cm-high edge made of clear Plexiglas surrounded the open arms to avoid falls. Each mouse was placed in the middle of the maze facing the open arm. Following 5 min of testing, mice were returned to their home cages. Arm preference was automatically analyzed using the Anymaze video tracking software (Stoelting, USA), and time spent in each arm and number of arm entrances were recorded. Elevated plus maze experiments were conducted under a light intensity of 1,300 lux to promote anxiety in the mice and apparatus was cleaned with ethanol between tests. PLOS ONE | www.plosone.org 2 October 2012 | Volume 7 | Issue 10 | e47522