Breeding. As noted earlier, the sdy autosomal recessive mutation arose spontaneously in DBA/2J mice at the Jackson Laboratory that were maintained as a closed breeding colony using obligate sdy/+ mice. The Jackson Laboratory later backcrossed sdy/sdy mice on the original DBA background with pure C57BL/6J mice for five generations, which were then intercrossed to obtain homozygous sdy/B6 mice. We again mated males of these sixth-generation homozygotes from the Jackson Laboratory with female B6 mice from the same source. Female and male sdy/+ offspring of this mating were then intercrossed to produce all the wild-type, sdy/+ and sdy/sdy mice tested in this study.
Genotyping and PCR. While sdy/sdy mice are readily idengified by their sand-colored fur, the coat color of sdy/+ mice is not sufficiently different from that of wild-type animals to allow reliable idengification. All animals were thus genotyped using a duplex polymerase chain reaction (PCR) procedure designed to yield PCR products across the segment of Dtnbp1 deleted in sdy mice (Li et al. 2003). The primers for the wild-type gene, yielding a PCR product of 472 base pairs, were SE3R (5′-AGCTCCACCTGCTGAACATT-3′) and SE3F (5′-TGAGCCATTAGGAGATAAGAGCA-3′). The primers for the sdy gene, yielding a product of 274 base pairs, were Sandy Forward (SF) (5′-TCCTTGCTTCGTTCTCTGCT-3′) and Sandy Reverse (SR) (5′-CTTGCCAGCCTTCGTATTGT-3′). The 472-base-pair product is detected only in wild-type and sdy/+ mice, while the 274-base-pair product is detected only in the sdy/+ and sdy/sdy mice. The 472-base-pair product is not detected in sdy/sdy mice.
Husbandry. The present study was conducted on (3 genotypes) × (2 sexes) = (6 groups) of 10–11 mice each. Mice were housed in same-sex cages, five per cage and maintained in a temperature/humidity controlled room under circadian light cycle of 12 h light/dark with the light cycle beginning at 0700 h. Mice had access to food and water ad libitum. Mice were weaned at postnatal day 21, and all testing began when mice were 3–4 months of age. Animals were allowed to acclimate to testing rooms for 1 h prior to testing. All behavioral tests were performed between 1030 h and 1800 h. The animals had never experienced any form of behavioral testing prior to the studies described here, and the order of the testing was designed to proceed from the least stressful to the most stressful tests. While behavioral testing was conducted blind to genotype for the wild-type vs. heterozygous sdy/+ mice, the pale coat color of homozygous sdy/sdy mice precluded completely blind testing. All procedures related to animals were performed in accordance with University of Pennsylvania Institutional Animal Care and Use Committee and University Laboratory Animal Resources policies and guidelines.
Experimental behavioral procedures
Neurological tests. The neurological screen was adapted from the Irwin screen (Irwin 1968), which has been widely used for testing neurological toxicity of drug candidates by pharmaceutical companies. This neurological screen is similar to phase 1 of the SHIRPA screen (Rogers et al. 2001). The mouse was weighed, then placed into an empty cage and observed for 3 min. A number of physical characteristics, including poor grooming, bald patches, absence of whiskers, labored breathing and blood around the nostrils, were characterized and recorded. Several behavioral responses were assessed (i.e. jumping, sniffing, rearing, approaching an object, movement throughout the cage, and urination and defecation). Visual acuity was assessed by placing the animal on a visual cliff platform (28 cm from the ground) for 2 min. Behavioral responses were recorded (i.e. approach to edge and poking nose over the edge). Lastly, sensorimotor reflexes (stabilization, righting, eye blink, ear twitch and whisker touch) were evaluated and recorded. The animals’ behaviors during the neurological test were scored qualitatively in real time by the same investigator.
Open-field exploration. Spontaneous locomotor activity was evaluated using the open-field exploration test 24 h after neurological tests. The square opaque white Plexiglass apparatus [40 × 40 × 32 cm (Everything Plastic, Philadelphia, PA, USA)] was cleaned prior to testing and between animals with 95% ethanol. A video camera was mounted directly above the apparatus. The field was divided virtually into two regions of interest: a center area (15 × 15 cm) and a peripheral area (Viewpoint VideoTrack version 2.0, Champagne Au Mont D’or, France; see Behavioral Analysis). A lamp with a 60-W bulb approximately 1.92 m away and 1.40 m above the apparatus was the sole source of illumination, providing an illumination intensity of 7 lux in the center of the field. The animals were placed in the center of the open field and given two 5-min sessions, 35 min apart. Distance traveled and time spent in the brighter center (which tends to be aversive to mice) and the peripheral areas during the two 5-min sessions were recorded automatically using the Viewpoint tracking system.
Rotarod. Motor co-ordination and balance were assessed using an accelerating rotarod (San Diego Instruments, Inc., San Diego, CA, USA) 24 h after the open-field test. Mice were first habituated to the rotating rod at a constant speed of 4 r.p.m. for 300 seconds (5 min), during which time latency to fall was not recorded. The habituation trial was only performed on the first day. If a mouse fell off, it was quickly placed back on top of the rod until the 5-min trial was completed. Mice were subsequently exposed to a rotating rod starting at 4 r.p.m. and linearly accelerated to 40 r.p.m. over a 5-min period. Three trials were administered per day with a maximum time of 300 seconds (5 min) and a 30-min inter-trial rest interval for 5 days consecutively. Performances in the daily trials were averaged for data analysis. Animals were tested at approximately the same time each day.
Elevated zero maze. Anxiety-like behavior was assessed using the elevated zero maze 24 h after the rotarod. The maze is a circular platform (62 cm wide) consisting of two opposing open and closed quadrants. A lamp with a 60-W bulb approximately 1.92 m away and 1.4 m above the maze was the sole source of illumination, providing an illumination intensity of 7 lux from the center of the maze. The maze was cleaned prior to testing and between animal tests with 95% ethanol. A video camera was mounted directly above the maze. Mice were placed in one of the closed quadrants and activity was recorded automatically for 5 min using the Viewpoint VideoTrack version 2.0 (see Behavioral Analysis).
Morris water maze. 24 h after the elevated zero maze, spatial learning and memory were assessed with the water maze as described by Morris (1984) and adapted for mice. The mouse water maze consisted of a circular pool (1.2 m diameter and 36 cm high), filled to a depth of 17 cm. The pool circumference was divided into four virtual quadrants and arbitrarily marked with the start positions: north (N), south (S), east (E) and west (W). The water was opacified with white non-toxic Crayola paint. The water temperature was between 22°C and 24°C during testing. The platform was made of transparent Plexiglas (10 cm × 10 cm), and its placement in the N quadrant of the pool remained fixed throughout training. The top of the platform was 1 cm below the surface of the water. The pool was surrounded by various distal spatial cues and illuminated by two lamps. All trials were recorded with a video camera (see Behavioral Analysis) situated above the pool. To prevent hypothermia, trials were separated from each other by a period of 20 min. Each animal rested on warming mats between trials.
Pretraining. Before the first trial, each animal was placed on the visible platform whose submerged location was made visible by an attached flag as a proximal cue for 10 seconds. The animals were then placed in the water and allowed to swim for 10 seconds before being guided back to the platform, where they were allowed to rest for 10 seconds.
Visible training: Each trial began by placing the animal in the water at the edge of the pool facing the wall from different quadrants. During the trial, each mouse was allowed 60 seconds to locate the platform. If the mouse failed to reach the fixed platform in the allotted time, it was guided to the platform. The start locations sequence was S, W and E. Each mouse was given two blocks of three trials for two consecutive days.
Hidden training: The flag was removed from the platform. Each mouse was placed in the water and allowed 60 seconds to locate the platforms. It was given two blocks of three trials daily until the wild-type animals were able to locate the fixed platform in 15 seconds or less. The start locations sequence was S, W and E. Hidden platform training was performed for 5 days with two blocks of three trials.
Probe trial: On the final day of hidden training, the platform was removed from the pool, and the mouse was placed in the quadrant directly across from where the platform had been. Each animal was allowed 60 seconds to search the pool. At the end of the trial, the mice were removed by hand.
During the platform trials, latencies (the time to reach the platform from the start location) were measured. Data from trials/day were averaged. In the probe trials, the performance measures were the mean swim speed, percentage of time spent in the each quadrant and the number of annulus crossings (number of times an animal crossed the exact place where the platform had been located during training).
Data were analyzed with a statistical software package, GraphPad Prism 5.0 for Windows, (San Diego, CA, USA) and JMP6 (SAS, Cary, NC, USA). Normality of the data for each variable was determined using the D’Agostino and Pearson (1973) test. One-way analysis of variance (anova) was used for analysis of weight. For analysis of the open-field data, the distance variable could not be considered normal and was not transformable to be sufficiently close to normally distributed to allow application of normal-based statistical approaches. Therefore, to compare the open-field distance across genotype and sex, a non-parametric version of a repeated measures anova was used, as described in Brunner et al. (2002) The model used (denoted F2_LD_F1 in the book) allowed for three factors, two non-repeated (sex and genotype) and one repeated (time). For analysis of the data from rotarod, elevated zero maze and Morris water maze, repeated measures anovas and one-sample t-tests were used. All analyses compared group differences by genotype, sex and genotype × sex interactions. All figures display the mean ± SE. For all analyses, significance was defined as P ≤ 0.05.