Developmental Psychobiology fuller understanding of the ontogeny of anxiety-like behavior in male and female rodents will provide the basis for future investigations of the link between hormones and anxiety. METHODS Subjects and Housing The subjects were 72 (35 males and 37 females) Lister-hooded rats (Rattus norvegicus) selected from nine litters bred in-house (breeding stock acquired from Harlan, Blackthorn, UK). The breeders and offspring were housed in a holding room (lights on 07:00–19:00; temperature: 20 1 C; relative humidity: 55 5%) in opaque plastic and wire mesh home cages (52 cm 40 cm 26 cm). Water and soy-free pelleted food were available ad libitum. The subjects were weaned into same-sex groups at pnd 21 and housed in same-sex sibling pairs or triplets at pnd 29. Different groups of animals were used in each age category, as prior exposure to novel apparatus has been shown to inﬂuence later performance (e.g., Bertoglio & Carobrez, 2000). The following numbers of subjects were allocated to each age category: adolescence (pnd 34–39) ¼ 9 males and 10 females; late adolescence (pnd 51–55) ¼ 9 males and 8 females; young adulthood (pnd 65–69) ¼ 9 males and 10 females; and older adulthood (pnd 104–109) ¼ 8 males and 9 females. No more than two subjects of each sex were taken from a single litter within each age group. Each animal was handled only for the purpose of transporting between the holding room and testing room, and for weekly weighing and cage cleaning. Apparatus The ET consisted of an area of vinyl ﬂoor (measuring 120 cm 120 cm) enclosed on all four sides by a wooden, painted wall (measuring 50 cm in height). A round, opaque plastic box (18 cm 14.3 cm) was placed mid-way along a wall of the arena with a single entrance/exit hole (8 cm 7 cm) directed toward the center of the arena. At the beginning of the test, the subject was placed inside the box via a lid. The OF was identical to the enclosure described above, minus the box. The ﬂoor of the arena was marked into nine areas (eight outer and one central area) by four lines, each 30 cm from one of the walls. At the beginning of each test, the subject was placed into the front left corner of the arena. The EPM consisted of four painted, wooden arms (51 cm 11 cm) raised 56 cm from the ground on a metal frame. Two of the arms had gray wooden walls (closed arms; 40 cm high) and the remaining two arms lacked walls (open arms), and the central area was 11 cm2. At the start of the test, the subject was placed into the central area facing a closed arm. Experimental Design The subjects completed the three tests in the following order: ET, OF, and EPM. All subjects received the tests in the same order, so Ontogeny of Anxiety-Like Behavior in Rats 733 that any possible order effects were uniformly distributed across age groups and variance between individuals on each task was minimized. The animals completed one test per day for three consecutive days, and all tests were conducted between 11:00 and 15:00 hr in the same testing room under dim, white light (approximately 25 lux). The apparatus was surrounded by a black curtain to reduce the number of external cues visible to the subject, and a video camera attached to the ceiling relayed images to a computer. Immediately prior to a test, the subject was transported to the test room in a small, covered box. After each test, the subject was returned directly to the home-cage, and the apparatus was cleaned with a 70% alcohol solution. Each test lasted 10 min, during which the following behavioral data were collected either by Ethovision XT software (Noldus Information Technology, Wageningen, The Nether- lands) or by manual entry onto a laptop computer running in- house software. In the ET, the latency to exit the start box, the total number of times that the subject re-entered the box, and total peeks without exiting the box were recorded manually into the computer by the observer. A peek was deﬁned as a subject putting its head out of the start box, up to the level of the ears, without exiting. In the OF, the number of entries into a new area was recorded manually, and an entry was deﬁned as all four paws crossing a line. Total locomotion was calculated as the total number of line crossings; however, as line crossings have been suggested to be an unsuitable method for comparing locomotor activity across ages (Vila, Philpot, & Kirstein, 2004), Ethovision was also used to calculate total distance moved (TDM; cm) and percentage of time spent mobile. Rears were also manually recorded into the computer, with a rear being deﬁned as an animal sitting on its hindlegs and stretching itself upwards. In the EPM, the percentage of time spent on the open arms and the percentage of open arm entries (relative to total line crossings) were recorded manually, in addition to rears and peeks onto the open arms (as deﬁned in the ET and OF). Ethovision also provided information on the TDM and the percentage of time spent mobile in the EPM. Statistical Analyses The behavioral data were checked for normality using the Kolmogorov –Smirnov test. Data not normally distributed were subject to a log transformation. Normal and normalized data were analyzed using multivariate analyses of variance (MANOVA) with age and sex as between-subject variables and the above behavioral measures as within-subject variables. Variables not normally distributed were analyzed using Kruskal – Wallis tests. Planned post hoc trend tests (polynomial linear/quadratic contrasts) were performed to examine patterns of behavioral change from adolescence to adulthood. Where there was a main effect of age on locomotion measures (TDM and total line crossings in the OF), an analysis of covariance (ANCOVA) was performed using body weight as a covariate. Age sex interactions are only reported if signiﬁcant. Pearson’s correlation coefﬁcient tests were used to examine the relation- ship between measures on the behavioral tests. Body weight data were analyzed using a univariate ANOVA with age and sex as between-subjects variables. The data reported in parentheses are means standard errors (SEMs). SPSS Version 17.0 was used