PMID: 27688943 PMCID: PMC5036436 DOI: 10.1002/brb3.514
Materials and Methods
Treatment with sevoflurane
C57/BL6 mice were used throughout the study, which was approved by the SUNY Downstate IACUC. A total of nine litters of mice were used to establish the approximate MAC for P7 mice. A separate set of 11 litters of mice were used for treatment without tail clamp and mice from this group were used for behavioral tests later on in life. At P7, all male pups from each litter (ranging from 2 to 6 pups) were randomly assigned to either the sevo or the no sevo (control) group, while the female pups remained with the dam. During a 2‐h treatment period, pups from the sevo group were separated from the dam and exposed to sevo in a 40% oxygen (O2) and 60% nitrogen (N2) gas mixture (GTS‐WELCO, Newark Distribution, Morrisville, PA). These pups were placed on a 37°C heating pad to prevent hypothermia during treatment. A pulse oximeter sensor (MSTAT 4 mm, Kent Scientific Corporations, Torrington, CN) was placed on one of the hind paws of the pup and measurements for heart rate (HR) and blood oxygen saturation (SpO2) were recorded every 5 min. To establish the approximate MAC of sevo on P7 mice, each treatment of sevo consisted of two mice and tail clamp was done every 10 min. The sevo concentration was adjusted to a higher concentration if both mice moved during tail clamp, adjusted to a lower concentration when neither mouse moved during tail clamp, and no adjustment was made when only one mouse responded to stimuli. This was our approach to determine that with a given sevo concentration, 50% of mice did not respond to stimuli. The sevo concentration was recorded every 5 min since that was the time interval that we used to record peripheral capillary oxygen saturation (SpO2) and HR. The pups from the control group were also separated from the dam and exposed only to 40% O2 and 60% N2. At the end of the 2‐h treatment the pups were returned to their home cage and reunited with their dams. All pups were then reared and weaned following standard institution procedures.

Behavior tests
The mice that were involved in the behavior tests had undergone a 2‐h sevo or no sevo treatment at P7 without tail clamping. They were reared and group housed under standard conditions. The sevo‐treated mice were marked to distinguish them from the no‐sevo–treated mice within a litter. We examined at most one to two litters of mice at a time for each behavior, with at least 1 week of resting time in between different behavioral tests. The behavior tests were given sequentially for the active place avoidance (APA), reciprocal social interaction, and olfaction habituation/dishabituation. After completion of these tests, we then introduced three‐chamber interaction, open field, and novel object recognition (NOR). All behavioral apparatus were assembled and remained in their original locations throughout the entire duration of the project. The APA test was done on mice starting at the age of P27. All other behaviors were conducted on mice within the age range of 1.5–5 months old. The following reasons contributed to variation in the number of mice used for some tests. First, we were not able to examine all treated mice on the APA due to irreparable malfunctioning of the APA apparatus. Therefore, we introduced NOR as a second cognition test on mice that had not been used for the APA. Second, some mice were not included in testing if they were not within the age range at the time of the test, specifically the second group of tests such as three‐chamber interaction, open field, and NOR. Besides the APA and the open field, all other tests that required manual scoring were first videotaped and then scored by experimenters who were blind to the treatment status of the mice.

Locomotion and anxiety‐like behavior
Open field test An open field apparatus was used to assess the general physical and anxiety‐like performance of the mice based on their ambulatory locomotion in the arena (Crawley 1985). In a well‐lit novel room, each mouse was given 30 min to explore the open field arena. Locomotion activities such as distance and time in different compartments of the arena were automatically measured using a computerized tracking apparatus (Versadat, Versamax, Groovy, CA).
Learning and memory‐like behavior
Active place avoidance test The APA test is a hippocampus‐dependent spatial memory test. A rotating arena consisting of a circular platform (40 cm diameter) was placed in the center of a dimly lit room. The mouse was trained to avoid a 60° shock zone, which could be defined within a region of the room identified by multiple visual cues (Fenton et al. 1998; Wesierska et al. 2005). Two‐day trials were performed as described previously (Burghardt et al. 2012). Briefly, the mouse was given 10 min for each trial with at least a 50‐min intertrial interval. The locomotion of the mouse was tracked by computer‐based software that analyzed images from an overhead camera and delivered shocks appropriately (Tracker, Bio‐Signal Group Corp., Brooklyn, NY). A brief constant current foot shock (500 msec, 60 Hz, 0.2 mA) across pairs of rods was delivered to the shock zone upon entrance of the mouse. Track analysis software (Bio‐Signal Group Corp.) was used to compute the number of times that the mouse entered the shock zone.
Novel object recognition test This is a two consecutive day test examining learning and memory‐like behavior on adult male mice (3–5 months of age; Leger et al. 2013). The test was conducted in a room with dim lighting. Day 1 is considered the familiarization phase. Mice were individually habituated in a standard open field apparatus for 10 min. They were then taken out of the arena briefly and two identical glass bottles filled with pink silica gel were placed in the center of the arena. The glass bottles were positioned 5 inches from each other such that the mouse can travel freely across the center of the arena without obstruction. The mouse was then put back in the arena and allowed 10 min to become familiar with the two identical objects. Day 2 is the test phase. One of the glass bottles is taken out of the arena and replaced with a yellow laboratory tube rack (H 6.5, W 3.5, D 2 inches) as a novel object. The holes on the sides of the tube rack were taped to prevent the mouse from climbing on them during the experiment. The same mouse was placed in the arena for 10 min in an identical manner as day 1 and allowed 10 min of exploration time. The times spent sniffing and interacting with (attempting to climb up or jump on or at) the familiar and the novel objects were scored for each mouse.
Social interactions
Reciprocal social interaction The subject mouse (P7 sevo treated or no sevo control) was transferred from his home cage to a new cage with fresh bedding and allowed to habituate to the cage for 10 min. At the end of this 10‐min session, a novel male target mouse (that had not undergone treatment during P7) of similar age was introduced into the same cage. The subject and the target mice were allowed to interact for 10 min. The amount of time that the subject mouse spent interacting with the target mouse (push–crawl/following behavior), self‐grooming, and exploring the arena and the total time the mouse was mobile were scored manually (Silverman et al. 2010).
Three‐chamber interaction A three‐chamber apparatus made of clear plexiglass was used for this study (Nadler et al. 2004). The apparatus is divided into three equally sized compartments (H 9.5, W 8, D 16 inches). First, the subject mouse was habituated for 10 min in the center chamber. Then the doors that give access to the left and right sides of the chamber were opened allowing the subject mouse to freely explore all three chambers for 10 min. During this time, the novel target mouse was habituated under a wire pencil cup on a separate tabletop. After 10 min of three‐chamber exploration, the doors were closed and the subject mouse was briefly confined in the center chamber. During this time, we set up the three‐chamber apparatus such that the novel target mouse was placed on one side of the chamber and a novel empty pencil cup on the other side. A weighted plastic cup was placed on the top of each pencil holder to prevent the subject mouse from climbing on the top of it. The doors were then opened to allow the subject mouse to explore the three chambers for 10 min.
Communication
Olfaction habituation/dishabituation The mouse was transferred to a new cage containing a thin layer of fresh bedding and a hole for inserting a cotton tipped swab. After a 10‐min habituation period in the new cage, the mouse was presented with nonsocial and social odors. Each odor was presented for three consecutive times; the order of presentation was water, almond extract (1:100, Spice Supreme), orange extract (1:100, McCormick), mouse socials 1 and 2. The mouse social odors were taken by wiping in a zigzag pattern across the bottom surface of different cages for odors 1 and 2; each cage housed unfamiliar mice of the same sex and strain. Each presentation of odor lasted 2 min. The amount of time that the mouse spent sniffing the cotton swab, including nose poking, chewing, sniffing, and close proximity (2 cm) of the nose to cotton swab was scored (Silverman et al. 2010).
Statistical analysis
All statistical analysis was done using GraphPad Prism 5.0 (GraphPad, San Diego, CA). Data with one variable such as the open field and the reciprocal social interaction were analyzed by t test. Data with two variables such as the APA, the NOR, the three‐chamber interaction, and the olfaction habituation/dishabituation were analyzed by two‐way ANOVA, followed by Bonferroni posttests.