]>NBA5261S0197-4580(99)00016-010.1016/S0197-4580(99)00016-0Elsevier Science Inc.Fig. 1Mean (SEM) body weight values. ∗Significant age effect for data combined across group (ANOVA, p < 0.0001). Tukey pairwise notations for combined data: 25-month and 17-month significantly differ; 317-month and 27-month significantly differ. Sample sizes are listed in parentheses.Fig. 2Morris swim maze performance. The first group of mice received 20 trials (5 days) of hidden platform training (A). The second group received 28 trials (7 days) of hidden platform training (B). ∗Significant age effect (ANOVA, p < 0.05). The 90-s probe trial was subdivided into six 15-s epochs for the first group (C), and for the second group (D). +Significantly different from chance (25%) for data combined across age (one-sample t, p < 0.05). Mean (SEM) cued platform distance for the second group collapsed across 5 trials (1 day) of training (E), and for each training trial (F). Tukey pairwise notations: 15-month and 27-month significantly differ; 25-month and 17-month significantly differ; 317-month and 27-month significantly differ. Sample sizes are listed in parentheses.Fig. 3Psychomotor battery performance. Mean (SEM) tightrope suspension time (A), crossings (B), and transformed score (C) collapsed across three trials. ∗Significant age effect for data combined across group (Brown–Forsythe, p < 0.05). Mean (SEM) square open field activity during a 10-min exposure (D). ∗Significant age effect for data combined across group (ANOVA, p < 0.05). Tukey pairwise notations for combined data: 15-month and 27-month significantly differ; 25-month and 17-month significantly differ; 317-month and 27-month significantly differ. Sample sizes are listed in parentheses.Fig. 4Eye pathology correlation with Morris swim maze performance of 17- and 27-month 129/SvJ mice. Spearman’s rank correlation was not significant for mean distance to find the hidden platform (A) nor mean probe trial performance (B). However, this correlation was slightly significant (p < 0.05) for mean distance traveled to the cued platform (C). Gross eye pathology was ranked on a four-point scale ranging from 0 (no eye pathology) to 3 (severe eye pathology; see text).Table 1Two groups, each comprising three counter-balanced age cohorts of 129/SvJ mice, were subjected to both a psychomotor battery and Morris swim maze. The schedule of behavioral tests is listed along with the day of the test for each groupBehavioral TestGroup 1 (Weeks 1–2)Group 2 (Weeks 8–9)Swim MazePretrainingN/ADay 1 (5 trials)Hidden PlatformDays 1–5 (20 trials)Days 2–8 (28 trials)Probe TrialDay 5 (1 trial 6 epochs)Day 8 (1 trial 6 epochs)Cued PlatformN/ADay 9 (5 trials)Psychomotor BatteryTightropeDay 10 (3 trials)Day 10 (3 trials)Open FieldDay 16 (10-min exposure)Day 11 (10-min exposure)ArticlesAge-related psychomotor and spatial learning deficits in 129/SvJ miceJohn MHengemihleaJeffrey MLongaJenniferBetkeybMathiasJuckercDonald KIngrama*doni@vax.grc.nia.nih.govaLaboratory of Cellular and Molecular Biology, Molecular Physiology and Genetics Section, National Institute on Aging, Intramural Research Program, Gerontology Research Center, Baltimore, MD 21224, USAbDenison University, Granville, OH, USAcDepartment of Neuropathology, Institute of Pathology, University of Basel, Schonbeinstr. 40, CH-4003 Basel, Switzerland*Corresponding author. Tel.: +1-410-558-8180; fax: +1-410-558-8323AbstractThe 129 mouse strain has been widely used to construct mutations that model behavioral aging in humans. The current study found significant age-related declines in both psychomotor and swim maze performance of 5-, 17-, and 27-month-old 129/SvJ mice. However, the age differences in swim maze acquisition were inconsistent with poor performance in the probe trial which assesses spatial memory. This inconsistency may result from the high degree of genetic polymorphisms and age-related visual pathology which afflicts this mouse strain. Therefore, we concluded that 129/SvJ mice present a problematic model of mammalian cognitive aging and involve a risk for behavioral contamination in studies involving mutant mice derived from this strain.KeywordsAgingAnimal behaviorEye pathologyGeneticsMaze learningTransgenic mice1IntroductionThe use of transgenic and knockout murine models has expanded opportunities for examining complex behavioral processes and may be clinically relevant in establishing models of neurodegenerative disease states. Homologous recombination in embryonic stem (ES) cells has allowed the development of chimeric mice with stable, germ line transmission of heritable targeted mutations [28]. Homozygous F2 offspring carrying the mutation can be backcrossed for several generations to create a congenic line on a fixed genetic background suitable for behavioral testing. The behavioral phenotype of these genetically engineered mutant mice is influenced by the mutation but also reflects the interaction of background genes [4], compensatory developmental mechanisms [18], and epistatic interactions between genes [15]. Therefore, before conclusions can be reached about the effect of a targeted mutation on polygenic learning and memory processes, it is important to understand the endogenous behavioral phenotype of the host strain. Because complex behaviors are polygenic, the genetic background of the host strain may affect the behavioral expression of a single gene mutation [28].ES cells isolated from the 129 mouse strain show the highest success rate for germ line integration [22,23]. Consequently this inbred mouse strain has been widely used in the construction of mutant mice, including murine models of Alzheimer’s disease [19,31]. However, relatively little has been published about age-related behavioral changes in this strain that would be useful for assessing the impact of mutant genes. It is possible that the behavioral abnormalities seen in mutant mice derived from 129 ES cells arise from polymorphisms in the mutant 129 genetic background rather than from any exogenous genetic manipulation [4]. Because of potential behavioral contamination from these background or hitchhiker genes [3,5,7,14], there is clearly a need for improved behavioral characterization of wild type 129 mice.To the best of our knowledge, this is the first study of aging in wild type 129/SvJ mice. We attempt to determine the suitability of this inbred strain to studies on behavioral aging. This is important because most previous studies on behavioral aging in mice have used the C57BL/6 strain [9,10,13]. In the current study, 129/SvJ mice of three ages underwent a motor and cognitive assessment, specifically locomotor activity in an open field, strength and coordination in a tightrope test, and spatial learning ability in a Morris water maze. Because previous studies had indicated relatively poor performance of 129/SvJ mice in the water maze [20], we examined whether extended acquisition training (28 versus 20 trials) could enhance performance in this task. We also attempted to ascertain the influence of age-related gross eye pathology on water maze performance of this albino mouse strain [24,25]. Previous studies have also indicated that 129 mice may be especially vulnerable to handling stress [17]. Therefore, we attempted to habituate our mice to handling and thereby overcome some of the known stress related activity confounds of the swim maze, such as passive floating [30] and jumping.2Methods2.1AnimalsExperimentally naive male 129/SvJ mice were obtained from the Jackson Laboratory, Bar Harbor, ME at 5-weeks of age (JAX® Stock Number 000691; now designated as 129XI/SvJ). Over a period of 22 months, three cohorts of mice were acquired and maintained at the Gerontology Research Center (GRC). At the time of behavioral testing, mice were either 5, 17 or 27 months old. Mice were housed in groups of five in a 30 × 19 × 13 cm plastic cage with corncob bedding and had access to food (NIH formula 07) and filtered water ad libitum. Mice were maintained on a 12:12-h light:dark cycle. Lights were turned on at 0700. The room temperature of the vivarium was about 22°C and the relative humidity was about 48%.2.2Behavior testing2.2.1HandlingFor logistical reasons, mice were divided into two counterbalanced groups and behaviorally tested 8 weeks apart. Before testing, mice were systematically handled in an attempt to minimize the previously observed passive floating behavior of this strain in the Morris swim maze (unpublished observation). Each mouse received 10-min of daily handling by the same experimenter. This consisted of picking up each mouse and continuously grooming its fur with brushes of various textures. This was done each day for a period of 2 weeks before behavior testing. To facilitate identification, a sterile AVID® microchip (AVID Inc., Norco, CA USA) was aseptically placed subcutaneously (s.c.) into each mouse before behavior testing.2.2.2Apparatus and procedureThe testing area consisted of a black rectangular table (112 cm × 104 cm) elevated 64 cm from the floor. This table was surrounded by white curtains (183 cm long) and was surveyed by a video camera mounted 122 cm overhead. The area was illuminated with overhead fluorescent lights that provided adequate contrast for the camera. Locomotor activity was monitored using a Videomex® tracking system (Columbus Instruments, Columbus, OH USA) which measured the amount of distance traveled by the mouse during the test period. Before testing, mice were transferred from the vivarium to the testing room 30 min before testing to allow for acclimation. All testing was conducted in the same testing room by the same experimenter between 0730 and 1500 h in the order described in Table 1. 2.2.3Maze trainingThe Morris swim maze is a spatial learning task in which mice must learn to escape by swimming to a concealed, submersed platform within a tank. The maze was a black, circular plastic pool, 80 cm in diameter and 50 cm in height, filled with clear (i.e. non-opaque) tap water to a depth of 25 cm. Water was maintained at 21–22°C. A transparent circular platform, 7 cm in diameter, was fixed in the center of one quadrant of the maze, 1 cm below the water surface. The swim maze was always maintained in the same position in the room as were all visible extra-maze cues. A white cloth curtain was draped around all sides of the maze to allow for adequate video camera tracking of mice within the maze. Previous studies conducted in our laboratory determined that C57BL/6J mice learn the Morris swim maze significantly faster in the presence of multiple three-dimensional cues in close proximity to the maze (unpublished data). It was assumed that these same cues would also benefit 129/SvJ mice. Therefore, seven three-dimensional objects (soda can, toy mouse, floppy disk box, nondescript foam rubber cutout, origami crane, light bulb, rubber stopper) were anchored equidistantly around the top inside wall of the maze and served as cues in this study. The cues varied in shape, size, color, and were no larger than 15 × 15 cm. Because a curtain surrounded the tank, the mice were unable to view the experimenter at any time. Distance traveled (cm) to reach the platform was the dependent variable.As shown in Table 1, the second group of mice (but not the first) were administered five pretraining trials to familiarize them with the presence of a submersed goal and to minimize any potentially confounding effect of nonspatial kinesthetic strategies that the mice may employ to solve the task. During pretraining, all cues were removed, and a Plexiglas® insert was placed into the tank forming a 7-cm wide alley leading to the submerged platform. Each mouse in the second group was given a total of five 90-s trials to locate the platform starting at the opposite end of the alley.For the hidden platform trials, the cues were replaced, and a trial was begun by placing the mouse into a counterbalanced starting quadrant (either N, S, E, or W) facing the wall of the pool. The computer then began recording the distance traveled. Each mouse had a maximum of 90-s to swim to the platform, at which point activity tracking was terminated. When the mouse reached the platform, it was permitted to remain there for 30 s before being removed from the maze. If the mouse failed to find the platform within 90 s, it was placed onto the platform by the experimenter and was removed after 30 s. Following a trial, the mouse was returned to its home cage with free access to food and water between trials. Following completion of a trial for all mice, a 70-min inter-trial interval (ITI) was provided before the start of a new trial. All mice dried themselves off during this rest interval and did not seem to be fatigued. Each mouse received four trials per day; 1 trial per day from each of the four counterbalanced starting quadrants. The platform’s location remained constant throughout training for each mouse. As shown in Table 1, the first group of mice received 20 trials (5 days) of hidden platform training, whereas the second group received 28 trials (7 days). Acquisition was measured as mean distance traveled each day, i.e. each block of four trials, for each group of mice. Thus, a decrease in distance traveled over hidden platform trials implies an improvement in reference memory spatial learning processes.A probe trial was given to all mice following their final trial on the last day of training (see Table 1). The probe trial started from a position directly in the center of the tank and was identical to previously described trials, except that the goal platform was removed. The percent of total distance traveled in the training quadrant (the quadrant where the platform had been located throughout the experiment) was calculated for the entire 90-s trial. The probe trial was divided into six contiguous 15-s epochs, and the percent training quadrant distance was calculated for each epoch in an attempt to access extinction. The probe trial was used to determine the amount of spatial bias or habit strength that mice developed for the location of the platform. An increased spatial bias in the probe trial indicated that the mice had developed a memory for the spatial location of the platform and was an additional indicator of the degree of spatial learning improvement with training.As shown in Table 1, on the day following the probe trial, the second group of mice received five cued platform trials with a 70-min ITI. For this block of trials, the peripheral cues described above were removed. The submerged platform was restored to a quadrant 180° diagonal to its original location and was prominently cued by the insertion of a dowel stick with a green Styrofoam® ball (10-cm diameter) attached 15 cm above the water. The 129/SvJ strain is albino, and therefore subject to visual impairments that may confound a pure spatial learning interpretation of the swim maze task [27]. The cued platform trials can address this problem by serving to identify gross impairments in visual discrimination ability in addition to motoric and motivational impairments all of which are potential confounds of the swim maze.2.2.4TightropeStrength and motor coordination of each mouse was tested by its ability to grasp and to remain suspended from a rope (2 mm in diameter and 50 cm in length). The rope was stretched taut inside a white polyethylene tank (50 cm diameter × 30 cm high) which was filled with water (at 21–22°C) to a depth of 10 cm. During testing, each mouse was kept in a dry holding cage that did not contain any bedding material. Before the test began, the mouse was introduced to the water by holding it by the tail in the water for 5 s. For each of three subsequent trials, the mouse was raised by its tail above the rope and then lowered slowly until it grasped the center of the rope with both front paws. The body of the mouse was then slowly lowered below the rope and released so that the animal had to support its body weight by its grip or fall into the water 20 cm below. The suspension time until a fall into the water was recorded (60-s maximum); the clock was stopped when the mouse entered the water (not when it lost its grip). In addition, the rope had pen markings at 5 cm intervals along its length to measure the amount of horizontal movement (crossings) which occurred while the mouse was suspended (a crossing was counted when the animals leading front paw touched the marking). When the mouse fell into the water, it was immediately removed to the holding cage and allowed to rest for 30-s before the next trial. The suspension time and number of crossings were averaged across three trials. A transformed score was calculated for each mouse: Transformed Score = (mean suspension time) + 10 (mean number of crossings). Thus, an elevated transformed score indicates superior performance either in terms of grip strength or motor coordination or both.2.2.5Open fieldSpontaneous locomotor activity in a novel environment was assessed in this test. A black Plexiglas® square (40 cm wide × 17 cm high) with wire mesh (1.5-cm square openings) attached to one side was used to monitor total distance traveled during a 10-min exposure. The device was elevated 14 cm from the surface of a black table to allow for passage of waste. The apparatus was illuminated from above with fluorescent lights such that the center of the square received approximately 9 foot-candles whereas the corners received 5 foot-candles of light. Mice were allowed to acclimate to the test room for 30 min before testing. Ten seconds after the mouse was placed at the center of the square, distance tracking began and was measured for 10 min. The apparatus was cleaned with 100% ethanol after each mouse was tested to remove both odors and boli. All alcohol vapors were thoroughly removed before the next mouse was tested. Two mice (5 and 17 months old) from the first group died following tightrope testing; open field data were not collected for these subjects.2.3Eye pathologyAt the conclusion of behavior testing, the second group of mice was examined for blepharoconjunctivitis. This ophthalmic condition can affect humans and is observed in several strains of mice, A/HeJ, BALB/cJ, CBA/J, and 129/J [24,25]. It is characterized either by a flaking hyperkeratosis of the eyelids or by excessive production of sebum-like material from the meibomian glands, and the incidence increases with advancing age. To assess the degree of pathology, we examined the eyeball presentation of each mouse and ranked the observations on a four-point scale as follows: 0 = no detectable eye involvement; 1 = mildly swollen eyelid; 2 = moderate or bulging eye; 3 = severe periorbital abscess. No attempt was made to determine the etiology of the ocular pathology nor the relationship between this pathology and visual acuity.2.4Statistical analysisBody weight, tightrope and open field data were first analyzed using a 2(Group) by 3(Age) analysis of variance (ANOVA; BMDP 7D) to determine the appropriateness of combining the two separately tested groups of mice for statistical analysis. If the main effect of group was not significant, data were collapsed across group, and a 3(Age) ANOVA was calculated. If the main effect of age was significant, the Tukey Studentized Range method (with harmonic mean adjustment for unequal sample size) was used to determine individual differences between age groups. If Levene’s test revealed heterogeneity of variance between the groups, the Brown–Forsythe equality of means test [1] was used in lieu of ANOVA because it does not assume that the group variances are equal. Critical values for the BrownForsythe test were obtained from the F distribution with a loss of degrees of freedom to allow for unequal group variances. To determine the degree to which body weight affected tightrope performance, the Pearson correlation coefficient (r) was calculated.Swim maze data for each group were analyzed separately. Hidden platform data for the first group of mice was analyzed using a 3(Age) by 5(Block) ANOVA with repeated measures (ANOVA-RM; BMDP 2V) on the last factor—mean swim distance for each block of four trials. Likewise, hidden platform data for the second group of mice was analyzed using a 3(Age) by 7(Block) ANOVA-RM. Probe trial data for each group of mice were analyzed using a 3(Age) by 6(Epoch) ANOVA-RM on the last factor—percent total distance in the training quadrant for each 15-s epoch. Probe trial data for each group were also analyzed using a one-sample t-test to determine if the observed spatial bias was significantly above chance level (25%). In addition, probe trial data for each quadrant were separately analyzed using a 3(Age) ANOVA collapsed across group. This analysis was conducted to verify selective searching in the training quadrant. Cued platform data for the second group was analyzed using a 3(Age) by 5(Trial) ANOVA-RM on the last factor—swim distance for each cued platform trial. In all cases, significant F tests were followed by pairwise comparisons using the Tukey method. If Levene’s test was significant for any repeated measure, a variance stabilizing square root transformation was conducted.The relation between eye pathology and swim maze performance was determined using the Spearman rank-order correlation (rs; BMDP 3D). This coefficient was selected because exact numerical values could not be assigned to gross eye pathology rankings.3Results3.1Body weightThe body weight of each mouse was taken before the start of behavior testing. Data were collapsed across the two separately tested groups of mice and a 3(Age) ANOVA was computed. As shown in Fig. 1,  this analysis revealed a significant main effect of age (F(2,70) = 13.41, p < 0.0001). Pairwise mean comparisons using the Tukey method showed that 17-month-old mice weighed significantly more than either 5- or 27-month-old mice (p < 0.01).3.2Morris swim mazeThe hidden platform data for each group of mice was analyzed separately to ascertain the influence of extended training (see Table 1). Because Levene’s test was significant, a variance stabilizing square root transformation was conducted on both groups of hidden platform data. Fig. 2A  presents 20 trials of data for the first group of mice. A 3(Age) by 5(Block) ANOVA-RM showed a significant main effect of age (F(2,33) = 6.56, p < 0.01). Likewise, Fig. 2B presents 28 trials of data for the second group of mice. A 3(Age) by 7(Block) ANOVA-RM revealed a significant main effect of age (F(2,34) = 7.35, p < 0.01). Tukey pairwise mean comparisons were used to access age differences at different time points (see Fig. 2). Both groups of mice demonstrated appreciable learning across trials as evidenced by the significant main effect of block (Group 1: F(4,132) = 15.26, p < 0.0001; Group 2: F(6,204) = 6.99, p < 0.0001). The significant interaction between block and age in each group reflects impaired learning among the 27-month-old mice compared to the 5- and 17-month-old mice in each group (Group 1: F(8,132) = 2.87, p < 0.01; Group 2: F(12,204) = 2.70, p < 0.01). When examining the data collected during extended training provided to the second group of mice, two salient points emerged. First, no age group improved their performance when training was extended from 4 to 7 days. Second, the 27-month-old mice showed significant deterioration in performance during extended training.On their last day of hidden platform training, both groups of mice were administered a 90-s probe trial consisting of six 15-s epochs. Fig. 2C presents probe trial data (spatial bias for training quadrant) for the first group of mice which received 20 trials of hidden platform training; Fig. 2D presents probe trial data for the second group of mice which received 28 trials of hidden platform training. A 3(Age) by 6(Epoch) ANOVA-RM did not reveal a significant main effect of age, epoch nor interaction for neither group. One-sample t-tests revealed that when data were collapsed across age, the first group of mice performed at or slightly below chance level (25%) for each 15-s epoch (see Fig. 2C). However, the same analysis revealed that the second group of mice, which received extended hidden platform training, performed significantly above chance level in all but the first epoch of the probe trial (see Fig. 2D). Thus, despite not showing improved acquisition learning with extended training trials as measured by swim distance, probe trial memory for the location of the platform was improved with extended training, although no significant age differences were evident using percent quadrant distance as the measure of memory. Moreover, the relative probe trial performance was not great considering that levels of 35% to 40% would be clear evidence of a spatial bias. Based on an analysis of distance swam in each quadrant in the combined groups, no significant spatial bias was observed in any maze quadrant. In addition, there were no significant age differences observed in any of the other quadrants. The percent distance scores for other (i.e. non-platform) quadrants were as follows: 5-month group (23.5%, 25.3%, 22.7%), 17-month group (24.2%, 24.4%, 24.8%), 27-month group (25.2%, 23.4%, 27.1%).The second group of mice was given five cued platform trials on the day following the probe trial (see Table 1). As shown in Fig. 2E, when the data were collapsed across all five cued trials, a 3(Age) ANOVA was not significant. As shown in Fig. 2F, when the data were analyzed as a function of training trials, a 3(Age) by 5(Trial) ANOVA-RM did not reveal any significant main effects nor interaction. There was an apparent age-related increase in distance to find the cued platform; however, this trend was not significant.3.3Psychomotor batteryBoth groups of mice were tested on the tightrope task following maze training (see Table 1). Data from the two separately tested groups were combined. Because Levene’s test revealed heterogeneity of variance between groups, the Brown–Forsythe statistic (F∗) was used. As shown in Fig. 3A,  a 3(Age) Brown–Forsythe test on the tightrope suspension time did not show a significant main effect of age. However, as shown in Fig. 3B, a 3(Age) Brown–Forsythe test on tightrope crossings revealed a significant main effect of age (F∗ (2,55) = 15.51, p < 0.0001). Pairwise mean comparisons using the Tukey method showed that 5-month-old mice made significantly more crossings than either 17- or 27-month-old mice (p < 0.01). Fig. 3C presents transformed data from the tightrope task for which a 3(Age) Brown–Forsythe test revealed a significant main effect of age (F∗ (2,55) = 4.43, p < 0.05). Tukey pairwise comparisons showed that 5-month-old mice performed significantly better than 17-month-old mice (p < 0.05).Interpretation of the age differences in the tightrope task were complicated to some degree by differences in body weight. Previous studies in our laboratory have shown that heavier mice tend to perform somewhat more poorly on the tightrope task [8,12]. As evidence of possible extraneous variation in the present study, the correlation between body weight and the transformed score for all age groups was significant, r (71) = −0.27, p < 0.05. However, this correlation was significant only within the 27-month-old group, r (16) = −0.48, p < 0.05. The significant decline in the tightrope transformed score between 5- and 17-month-old groups could be related to the heavier body weight of the 17-month-old group. However, as shown in Fig. 1, there was no significant difference in body weight between the 5- and 27-month groups to account for the decline in performance of this task.Both groups of mice were tested in the square open field (see Table 1). Data from the two groups were combined. As shown in Fig. 3D, a 3(Age) ANOVA revealed a significant main effect of age (F(2,68) = 5.50, p < 0.01). Pairwise Tukey comparisons showed that 27-month-old mice were significantly less active than either 17- (p < 0.05) or 5-month-old mice (p < 0.01).3.4Eye pathologyThe second group of mice was examined for gross eye pathology at the completion of behavior testing. Only 6% (1:18) of the 5-month-old mice were observed to have moderate to severe eye pathology suggesting that this cohort was relatively healthy. In contrast, 46% (6:13) of the 17- and 67% (4:6) of the 27-month-old mice were observed to have an eye pathology ranking of 2 or 3. Because the 5-month-old cohort was relatively healthy, their data were culled from the eye pathology correlation; only data from the 17- and 27-month-old cohorts were included because these mice presented most of the overt eye pathology. As shown in Fig. 4A and B,  the Spearman rank correlation with eye pathology was not significant for hidden platform distance (mean of first 20 trials) nor probe trial performance (mean of 6 epochs). However, as shown in Fig. 4C, a significant Spearman correlation was found between eye pathology and distance traveled to the cued platform (mean of 5 trials; rs (17) = 0.46, p < 0.05).4DiscussionEvidence of behavioral aging in male 129/SvJ mice was observed in tests of psychomotor performance and spatial learning in this cross-sectional study. Specifically, age was associated with diminished strength and motor coordination as measured by tightrope performance and decreased spontaneous locomotor activity measured in the open field. Significant age-related deficits in the water maze were evident in this strain of mice during the acquisition trials measured as distance to platform; however, there were no significant age differences in probe trial performance. The later observation would confound any conclusions about age differences in spatial memory abilities, despite the age differences in learning in this task.The behavioral phenotype of 129 mice is continuing to emerge in the literature. Previous studies have reported hypoactivity of 129/SvJ mice in the open field [15,17,18]. However, this robust finding was not reproduced in the current study. When compared to unpublished observations in 6-month-old male C57BL/6J mice from our own laboratory, the 5-month-old male 129/SvJ mice of the current study were found to be significantly (18%) more active in the open field. One previous study [18] using the tightrope task found no difference between C57BL/6J and 129/Sv mice. In contrast, the 5-month-old male 129/SvJ mice from the current study were found to demonstrate a significantly (36%) lower transformed score in the tightrope task when compared to 6-month-old male C57BL/6J mice (unpublished data). Methodological differences, such as the handling, may account for these discrepancies. However, it is more likely that the mixed genetic background of the 129/SvJ strain [26] accounts for many of these discrepancies with the previous psychomotor literature on 129/SvJ mice.The Morris swim maze is heavily dependent upon visual, motor, and cognitive abilities. As such, interpretation of a cognitive deficit in the current study is problematic from several perspectives. First, the observed age-related decline in psychomotor performance of this strain may confound a pure spatial deficit interpretation of the swim maze results. Second, although learning was evident in the two youngest groups, the aged group showed no significant improvement across sessions. Third, the level of performance achieved over 20 trials in 129/SvJ mice was substantially less than we have observed in young C57BL/6NIA [11] and C57BL/6J [2] mice as measured by distance traveled. Fourth, performance in the probe trial was poor for all three age groups and did not differ significantly. There was no evidence of spatial bias to indicate use of a spatial strategy based on utilization of visual cues. Although no significant age effect was found in the cued platform test, it was clear that many older mice seemed impaired as shown by the great variability. This variability was correlated with the degree of eye pathology. Over 52% (10:19) of the 17- and 27-month-old mice presented with overt eye symptoms, suggestive of blepharoconjunctivitis [24,25]. It is possible that the high incidence of age-related visual pathology in these mice may cause a visual acuity deficit resulting in a potentially confounding measurement error [9]. As the severity of the pathology increased, performance in the cued trials diminished (Fig. 4C). However, no such correlation was observed in the hidden platform task (Fig. 4A) or in the probe trial (Fig. 4B). These latter observations would indicate that the mice were not utilizing available visual cues to direct their performance in the swim maze. In summary, interpreting the observed age-related differences in spatial learning ability is very problematic to the extent that these learning deficits may not be a function of normal aging in 129/SvJ mice [9].As noted previously, the 5-month-old 129/SvJ mice in the current study presented a very low incidence of overt visual pathology. Therefore, their swim maze behavior was not likely to be influenced by the measurement errors discussed above. As such, the current study supports previous findings which showed young 129/SvJ mice achieved an inferior level of asymptotic performance in the swim maze relative to pigmented strains of mice [20]. Although 5-month-old male 129/SvJ mice from the current study were able to consistently find the hidden platform, this asymptote was only achieved with a highly significant (98%) increase in distance traveled compared to 7-month-old male C57BL/6NIA mice from our laboratory [11]. The current study demonstrated that without extended hidden platform training, 129/SvJ mice lack development of appreciable spatial bias as measured in the probe trial (see Fig. 2C); this finding replicates negative probe trial results of a previous study in 129/SvJ mice [20]. It is possible that the percent training quadrant distance measure frequently used in the probe trial is not a sensitive measure of spatial bias in 129/SvJ mice. Annulus crossings seems to be a better choice because another study [18] was able to demonstrate significant probe trial spatial bias in 129/Sv mice using that measure. The current study reports two manipulations which taken together were partially successful in improving probe trial performance. First, extended hidden platform training was able to improve spatial bias above chance performance (Fig. 2D). Second, our handling manipulation was apparently successful in reducing stress in that neither passive floating nor jumping behaviors were observed in any of our mice in the swim maze. This observation is significant because previous studies have shown that handling reduces stress and thereby improves spatial learning in mice [6]. Despite these relative improvements in spatial bias, however, probe trail performance did not vary significantly across age.Previous studies using aged rats have shown that a shortened ITI can impair water maze performance due to fatigue [21]. Therefore, we were careful to space the trials out in an attempt to avoid fatigue in our aged subjects. Absence of fatigue was evidenced by the fact that all the mice completely groomed themselves to dryness between trials and did not shiver. Moreover, the distance measure used in the current study to assess acquisition is more robust to fatigue-related activity confounds than the latency measure used in previous studies [18,20]. These previous studies have relied on distal room cues for navigation, however, in the current study proximal cues were placed inside the rim of tank. Because it has been suggested that mice are less adapted to swimming tasks than other rodent species [29], it is possible that this proximal location of cues favors the development of a spatial rather than kinesthetic strategy to solve the task.In conclusion, we feel that the high degree of documented genetic polymorphisms [26] and possible ancestral contamination [22] makes the 129/SvJ mouse strain generally unsuitable to studies of behavioral aging. Although we feel that the 129/SvJ strain should be avoided in experiments assessing age-related effects on behavior, we appreciate the value of this strain in the production of mutant mice. The strategy of maintaining the targeted mutation heterozygous (by backcrossing chimeras into a common inbred strain such as C57BL/6J) and then testing the homozygous mutant in an F1 cross is sound. Because F1s are used, specific deficits in the 129 strain influenced by deleterious homozygous genes will be attenuated. In addition, the current study reports visual pathology in aged 129/SvJ mice severe enough to potentially confound swim maze studies of aged mutant mice derived from 129/SvJ ES cells. Future studies using conditional mutants may avoid potential behavioral problems resulting from genetic variation and contamination [16].AcknowledgementsThe Gerontology Research Center (NIH-NIA-IRP) is fully accredited by the American Association for the Accreditation of Laboratory Animal Care. 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