Perinatal nicotine exposure and anxiety-like phenotype in elevated plus maze EPM. The percentage of time spent in the open versus closed arms A and the number of entries into open arms B of the EPM were analyzed. The behavioral task was initiated by placing the mouse at the center of the maze facing one of the open arms with free access to the entire maze.
Behavior was monitored via an overhead camera for a 5 min period. The time spent in the open arms and the closed arms, and the number of entries into open arms of maze were calculated from the video recordings. A rectangular Plexiglas box consisting of an exploration chamber 40 X 40 X 25 cm and a testing chamber 40 X 20 X 25 cm separated by a sliding door was used. On day 4, during the habituation phase, the mouse was placed in the empty exploration chamber for 5 min and then in the empty test chamber for another 5 min.
The transit from the exploration chamber to the test chamber was permitted by lifting the sliding door. If a mouse did not voluntarily enter the test chamber, it was gently guided to do so.
On day 5, during the acquisition session, the mouse was placed for 5 min in the exploration chamber containing five wooden objects of identical size but different shapes rectangle, triangle, circle, oval and octagon.
Next, the mouse was placed in the test chamber containing two wooden objects of the same size and shape circle for 5 min. The exploratory activity defined as interactions with the object s from a distance of not greater than 1. On day 6, the mouse was placed in the empty exploration chamber first and then in the empty test chamber, for 3 min each to permit habituation to the chambers. Next the mouse was returned to the exploration chamber, which now contained the same five objects that the mouse had explored on day 5.
The exploratory activity was recorded for 3 min. Next the sliding door was opened and the mouse entered the test chamber, which now contained two objects.
One object was of the same shape and size as one of the five objects the mouse had explored 3 min earlier in the exploration chamber familiar object , and placed in the test chamber in a position analogous to its position of the object of the same shape in the exploration chamber.
The mouse explored the two objects for 3 min, and exploratory activity was recorded. Preliminary analyses showed that the mice explored all four corners of the exploration and test chambers equally. Therefore, on days 5 and 6, the two objects were placed in the test chamber equidistant from the middle of the sliding door.
The position of the objects in the exploration and test chambers was fixed throughout the entire study. The familiar object was always a circle and the novel object a star throughout the entire study.
Since all mice met the inclusion criterion of exploring all objects in the exploration and test chambers on day 5, we did not have to exclude any mouse from the study. Initial analysis revealed that the time spent exploring the 5 objects by the mice in the three treatment groups was not significantly different.
All mice met this criterion, and therefore no mouse was excluded. A recognition index was calculated using the formula: On day 6 the mouse was placed for 5 min in the test chamber, which now contained two identical objects selected at random from a collection of 2 sets of identical objects: Two unopened and unmarked cans of food 3. The two sets are needed so that in the next stage of the test, a novel object can be drawn from a set to which the mouse was not previously exposed see below.
Total time spent exploring the objects as well as the time spent exploring the object placed on left versus right side was calculated. These data were used post hoc to evaluate object or side bias. A counter-balanced design was used to address potential bias.
Next, the mouse was returned to its home cage. Following 10 min in the home cage, the mouse was returned to the test chamber, which now contained one of the previously explored objects and a novel object chosen from the set not used in the previous step. The mouse was allowed to explore the objects for 5 min. The total time spent exploring both the objects as well as time spent exploring each object were calculated. During each stage of the test except during the 10 min stay in the home cage , the behavior of the mouse was recorded using an overhead video camera.
An investigator blinded to the identity of the mouse analyzed the video recordings. Based on an exclusion criterion of less than 30 sec exploration of objects i. The novel object recognition index was calculated using the formula: Cliff avoidance reflex CAR Impulsive-like behavior was assayed using an apparatus consisting of a custom-built round Plexiglas platform 20 cm in diameter supported on a plastic rod 50 cm in height similar to a barstool [ 23 ].
The mice were placed individually on the platform and their behavior was recorded via an overhead video camera for a period of 60 minutes. When a mouse fell off the platform, it was gently picked up and returned to the platform.
An investigator blinded to the identity of the mouse analyzed the video recording to calculate the average length of time to first fall latency to first fall during the 60 min interval. A total of 6 behavioral tests were conducted in this study with approximately one-week interval between the tests.
Using a mouse in every one of the 6 tests sequentially would have meant that the mouse would have been approximately 2 months older by the end of test 6 compared to their age during test 1.
Moreover, we were concerned that the experience of undergoing more than 3 behavioral tests consecutively could influence performance in the subsequent behavioral tests. To avoid the potential influence of these variables, one male and one female mouse from each litter were assigned to one of two groups. Each group of mice was tested in only one battery of behavioral tests. The NOR test was conducted in male mice only.
Statistical analysis We confirmed normal distribution of data from all our experiments. Therefore, differences between experimental groups were analyzed for statistical significance using parametric statistical tests. Two-way ANOVA was used to test the main effects of perinatal treatment and sex, and the interaction between these two factors.
Bonferroni multiple comparison post hoc test showed that the P90 male mice had significantly greater body weight compared to females, regardless of the perinatal treatment [ S1 and S2 Tables].
The average length of pregnancy 19—20 days , litter size at birth 6—9 , and sex ratio at birth 1: The following developmental milestones were achieved on average at the same time by all offspring in the three treatment groups: Ears detached on P4, fur appeared on P and eyes opened on days P None of the other pair-wise comparisons were significant [ S1 and S2 Tables].