PMID: 18667523 PMCID: PMC2721666 DOI: 10.1093/toxsci/kfn152
MATERIALS AND METHODS
Animals Sprague-Dawley dams (n = 48) had normal vaginal births and on the day of birth (PND 0), each litter was separated by sex, and four males and four females were randomly selected so that each litter was culled to eight. The dams with their natural litters (culled to four/sex/litter) were obtained on PND 0 from the breeding colony at the National Center for Toxicological Research (NCTR/FDA). Each dam was individually housed in a standard polycarbonate cage lined with wood chip bedding and provided with ad libitum food (NIH-31, Purina Mills, St Louis, MO) and water. The colony room was maintained at 22°C ± 1°C (mean ± SE) and 45–55% humidity on a 12-h light/dark cycle (7:00 a.m.–7:00 p.m.). Each pup was paw tattooed on PND 1 and also identified with a nontoxic marker on the dorsal side and tail tip on PND 4. All animal procedures followed the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996) and were approved in advance by the NCTR Institutional Animal Care and Use Committee.
Treatment Ketamine hydrochloride (100 mg/ml solutions as Ketaset, Fort Dodge Animal Health, Fort Dodge, IA) was diluted with saline to produce 2 mg/ml solutions. PCP (NIDA, Bethesda, MD) and l-carnitine (Sigma-Aldrich Corp., St Louis, MO) were dissolved in 0.9% saline. Ketamine hydrochloride (400 μl) and l-carnitine (500 mg) were diluted with 10 ml of saline to produce 40 mg/ml KET and 500 mg/ml l-carnitine solutions, respectively. These solutions were combined in a 50 ml conical tube to obtain the KLC dose (250 mg/kg l-carnitine and 20 mg/kg ketamine) injected on PND 7. Solutions were made weekly and kept refrigerated. The sc injections were done using a 25-gauge needle.
The within-litter treatment (one pup/sex/treatment/litter) was a particularly important aspect of the experimental design since it is well recognized that differences in maternal care can affect offspring behavior (Barron and Riley, 1985; Fleming et al., 1999) and, at least in rats, pup behavior determines some aspects of maternal care (Marino et al., 2002). Thus, similar to that described by Zissen et al. (2007), overall maternal care was controlled at the litter level in that each dam cared for a litter which contained pups of all treatment groups. However, as noted by Zissen et al. (2007), this cannot prevent or control for differential treatment of individual pups by the dam.

Treatment assignment was based on PND 4 body weight such that all groups had similar average body weights prior to treatment. The four groups were (1) 10 mg/kg PCP at 12:00 p.m. on PNDs 7, 9, and 11; (2) six injections of 20 mg/kg KET on PND 7 (8:00 a.m.–6:00 p.m.), separated by 2-h intervals; (3) six injections of 20 mg/kg KET and 250 mg/kg l-carnitine on PND 7 (8:00 a.m.–6:00 p.m.), separated by 2-h intervals followed by 250 mg/kg l-carnitine at 12:00 p.m. on PNDs 8–11; and (4) six injections of saline at on PND 7 (8:00 a.m.–6:00 p.m.), separated by 2-h intervals followed by saline at 12:00 p.m. on PNDs 8–11. The doses and treatment regimens were based on previous reports indicating that similar treatments caused neurodegeneration in rats (Ikonomidou et al., 1999; Scallet et al., 2004; Wang et al., 2001). The l-carnitine dose was based on studies of its protective effects against 1-methyl-phenylpyridinium ion–induced apoptosis (Wang et al., 2007). Thus, for each of the 48 litters, 1 male and 1 female were assigned to each treatment resulting in 48 pups/sex/treatment.

Body Weight Body weights of the offspring were recorded on PNDs 4, 7, 8, 9, 10, 11, and 18. On PNDs 8–11, body weights were recorded after behavioral testing and prior to treatment.
Home Cage Pup Behavior To determine the immediate effects of treatment, home cage behavior was assessed on PNDs 7–11. At each treatment time, the dam was placed in a holding cage. Each pup was then identified and when indicated, injected. Those pups not injected (e.g., PCP-treated pups at 8:00 a.m., 10:00 a.m., 2:00 p.m., and 4:00 p.m. on PND 7 and on PNDs 8 and 10 as well as the KET-treated pups on PNDs 8–11) were handled in a manner similar to the injected pups. Time of the last injection/handling for each litter was recorded, and the dam was returned to the home cage. Time from dam removal to replacement into the home cage was less than 120 s. At 5, 14, 23, and 32 min posttreatment, the behavior of each pup was assessed by one of two experimenters blind to treatment. Thus, there were four observations at five of the six treatment times on PND 7 (i.e., pups were observed after injections/handling at 8:00 a.m., 10:00 a.m., 12:00 p.m., 2:00 p.m., and 4:00 p.m., but not after the 6:00 p.m. injection/handling time). On PNDs 8–11, there were four observations following the 12:00 p.m. treatment time. Each pup was categorized as exhibiting one of 12 different behaviors (see Table 1) which were based on a previous scoring system (Goodwin and Barr, 2005). Only one behavior/pup/observation time was recorded.

Slant Board Behavior (Negative Geotaxis) Vestibular system integrity and Motor coordination were examined using a slant board test as previously described (Adams et al., 1985). Briefly, between 7:30 and 9:00 a.m. on PNDs 8–11, the dam was removed and each pup was placed on its ventral side with its nose pointing toward the lower end of a sandpaper-covered 45° incline board. Each pup was allowed 60 s to complete a 180° turn. One trial/day was conducted, and the latency to turn or fall from the apparatus was recorded by a tester blind to treatment conditions.
Forelimb Hang Behavior Muscle strength/coordination was examined using a forelimb hang test as previously described (Cada et al., 2000). Briefly, between 7:30 and 10:00 a.m. on PNDs 12–16, the dam was removed and each pup was placed on a taut string stretched between two blocks of wood spaced 46 cm apart and 41 cm above a padded surface. One trial/day was conducted, and the latency to fall was recorded (maximum 60 s) by a tester blind to treatment conditions.
Statistical Analyses
Body weight. Offspring body weights were compared using ANOVAs with factors of treatment (control, KET, PCP, and KLC), sex, and the repeated measure of PND (JMP, Version 7.0; SAS Institute Inc., Cary, NC). Tukey post hoc tests were used to further analyze significant main effects or interactions.
Home cage pup behavior. Data from the five observation times on PND 7 (8:00 a.m., 10:00 a.m., 12:00 p.m., 2:00 p.m., and 4:00 p.m.) were analyzed separately from the single observation time on PNDs 8–11 (12:00 p.m.). Six behaviors were categorized as abnormal activity: fast activity, paddling, partial paddling, paresis, partial paresis, wall climbing. To analyze abnormal activity, each pup at each observation at each time was assigned a “1” if it exhibited any of the six abnormal behaviors or a “0” for any other behavior. Generalized linear models with a log link and Poisson distribution were used to analyze the counts for each of the two data sets (PND 7 only and PNDs 8–11) with factors of treatment, observation time (e.g., 8:00 a.m., 10:00 a.m.) (PND 7 analysis only), minutes posttreatment (e.g., 5, 14, 23, or 32 min), and sex.
Slant board behavior. Each pup could exhibit one of three outcomes: a successful turn within 60 s, a fall from the apparatus within 60 s, or an incomplete turn. A failure was categorized as a fall or an incomplete turn. The odds of failure were analyzed using a generalized linear model with repeated measures and a binomial distribution and logit link function. To analyze the latency to turn time, a Cox Proportional Hazards model was run in SAS (SAS Version 9.1; SAS Institute Inc.) using treatment, sex, and PND as factors. Pups that fell or did not complete the turn were accounted for in this analysis by adjusting the empirical distribution function.
Forelimb hang behavior. To analyze the latency to fall, a Cox Proportional Hazards model was run using SAS (SAS Version 9.1, SAS Institute Inc., Cary, NC) with treatment, sex, and PND as factors.