Progressive and gender-dependent cognitive impairment in the APPSW transgenic mouse model for Alzheimer’s disease

Behavioural Brain Research 103 (1999) 145 – 162 www.elsevier.com/locate/bbr

Research report

Progressive and gender-dependent cognitive impairment in the APPSW transgenic mouse model for Alzheimer’s disease David L. King a, Gary W. Arendash a,*, Fiona Crawford b, Troy Sterk a, Julian Menendez a, Michael J. Mullan b a

Alzheimer’s Research Laboratory, Department of Biology, Uni6ersity of South Florida, Tampa, FL 33620, USA b Roskamp Institute, Department of Psychiatry, Uni6ersity of South Florida, Tampa, FL 33620, USA Received 12 October 1998; received in revised form 01 February 1999; accepted 03 February 1999

Abstract To determine if early cognitive/sensorimotor deficits exist in APPSW transgenic mice overexpressing human amyloid precursor protein (APP), Tg + and Tg − animals at both 3 and 9 months of age (3M and 9M, respectively) were evaluated in a comprehensive battery of measures. The performance of all Tg + mice at both ages was no different from all Tg− controls in Y-maze alternations, water maze acquisition, passive avoidance, and active avoidance testing. By contrast, results from other tasks revealed substantive cognitive deficits in Tg + mice that were usually gender-dependent and sometimes progressive in nature. Between 3M and 9M, a progressive impairment was observed in circular platform performance by Tg + males, as was a progressive deficit in visible platform testing for all Tg + animals. Other transgenic effects included both impaired water maze retention and circular platform performance in 3M Tg + females; this later effect was responsible for an overall (males + females) Tg + deficit in circular platform performance at 3M. Sensorimotor testing revealed several Tg + effects, most notably an increased activity of Tg + males in both open field and Y-maze at 3M. Significant correlations between a number of behavioral measures were observed, although factor analysis suggests that each task measured components of sensorimotor/cognitive function not measured by other tasks. Finally, Tg + mice had lower survivability than Tg − animals through 9M (85 vs. 96%). In summary, these results demonstrate the presence of gender-related and progressive cognitive deficits in APPSW transgenic mice at a relatively early age (i.e., prior to overt , b-amyloid deposition in the brain), suggesting a pathophysiologic role for elevated levels of ‘soluble’ b-amyloid in such impairments. © 1999 Elsevier Science B.V. All rights reserved. Keywords: APP transgenic mice; Cognitive deficits; Progressive; Gender-dependent

1. Introduction Insights into the etiology and progression of Alzheimer’s disease (AD), the most prevalent age-associated dementia, may be significantly advanced by transgenic animal models for the disease. The most notable transgenic models involve CNS overexpression of human amyloid precursor protein (APP) [13] or overexpression of mutated APP forms [‘Swedish’ mutation (APPSW), PD-APP] found in familial AD [11,16– * Corresponding author. Tel.: +1-813-974-1584; fax: + 1-813-9743263. E-mail address: [email protected] (G.W. Arendash)

18,20,29]. These mouse models have been shown to exhibit at least some AD neuropathology, such as: 1) a progressive increase in brain Ab levels, 2) diffuse and/or compact b-amyloid (Ab) deposition, and 3) an age-related increase in neuritic plaques (diffuse and/or compact). Most recently, transgenic mice bearing both the APPSW mutation and a presenilin 1 (PS1) mutation have been reported to experience an earlier elevation in brain Ab levels and earlier neuritic plaque development than mice with the APPSW mutation alone [4,14]. Although no neuronal loss or paired helical filament (PHF) formation has been observed in any of the above transgenic lines [17,18], their increased brain Ab levels, brain Ab deposition, and neuritic plaque formation are

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encouraging for further elucidation of neuropathologic similarities to AD. Unfortunately, the promise of transgenic AD models has thus far been diminished by a generally inadequate characterization of age-associated behavioral changes. Aside from the fact that very few studies have investigated multiple behavioral time-points in an attempt to establish progressive behavioral changes, the tasks used to evaluate cognitive performance in transgenics have been extremely limited and potential gender differences in performance have been essentially ignored. To date, nine studies involving APP transgenic mice have addressed behavior [6,9,14– 16,23,24,26,32], with only two of these studies presenting data on both cognitive performance and sensorimotor function [9,24]. All nine studies have relied, often exclusively, upon the water maze and/or Y-maze tasks. Although the majority of these studies have reported impaired cognitive performance of APP transgenic animals, only two studies presented evidence for progressive, age-related impairment (i.e., significantly poorer performance of transgenic mice tested at a later time point compared to those tested at an earlier time point). In transgenics overexpressing normal human APP751, Moran et al. [24] found decreased spontaneous alternation of transgenic animals tested in the Y-maze at 12 months of age (12M) compared to those tested at 6M. In transgenics overexpressing mutant human APP670,671 (APPSW), Hsiao et al. [16] reported deficits in water maze acquisition for transgenic animals tested at 9 – 10M, but not those tested at 3M or 6M. The APPSW transgenic mouse carries multiple copies of a doubly mutant human APP at sites 670/671, which is highly associated with early-onset (familial) AD. APPSW presents accretionary overexpression of APP in the mouse brain, as well as progressive increases in both Ab levels and neuritic plaque densities in the brain [14,16,31]. It is important to note that levels of soluble Ab are elevated in APPSW transgenics by 6 – 8M without substantive Ab deposition or neuritic plaque formation [14,16]. By 10 – 16M, however, Ab-containing neuritic plaques are present in brains of APPSW animals [10,16]. In APPSW transgenic mice aged 16 months, Irizarry et al. [18] found a 4 – 8% Ab burden in several cognitively-important brain areas — a percentage that is similar to that reported for human AD brains. Similar to neuritic plaques in AD, recent studies have demonstrated the presence of activated microglia in neuritic plaques of APPSW animals [10], as well as staining of APPSW plaques for markers of oxidative stress [27,30]. As with other transgenic models, behavioral changes have been less exhaustively documented in the APPSW transgenic mouse. Hsiao et al. [16] found that 10M APPSW mice alternated significantly less than control animals in the Y-maze and exhibited an impair-

ment in water maze acquisition that was not present at earlier test points. More recently, Holcomb et al. [14] determined that APPSW transgenic mice and doubly transgenic APPSW + PS1 mice were impaired to an equal extent in Y-maze spontaneous alternation at 3– 4M. Because of its robust neuropathologic characteristics and the aforementioned initial reports of behavioral deficits, the APPSW transgenic mouse was chosen for a thorough examination of sensorimotor capabilities and cognitive faculties in the present study. Transgenic and non-transgenic animals at both 3M and 9M were evaluated for effects of transgene, gender, and age on the comprehensive battery of measures selected. All behavioral measures in this study were collectively subjected to correlation analyses and factor analysis, with survivorship also being determined through 9M. Our findings indicate the presence of sexrelated cognitive deficits in APPSW transgenic mice, several of which are progressive in nature. Since these cognitive deficits occur prior to overt Ab deposition in the brain, the results indicate that elevated brain levels of soluble Ab are sufficient to produce behavioral impairments before substantive neuritic plaque formation.

2. Methods

2.1. Animals Fourteen healthy C57B6 females (Jackson Labs., Maine) and one male Tg2576 (APPSW) transgenic mouse (heterozygous C57B6/SJL F2, from McLaughlin Res.) were used to generate a group of 92 experimental animals. The mutant human transgene present in the founder male has been associated with familial Alzheimer’s disease and contains a double mutation (Lys670 “ Asn, Met671 “ Leu) of the amyloid precursor protein (APP695) [25]. The mutant gene has been shown to be responsible for an approximately 5-fold overexpression of APP695 in the brains of mice over 2M [16]. Correspondingly, b-amyloid is significantly elevated in the brains of 6–8M Tg2576 mice [14,16]. For this study, 50 3M mice and 42 9M mice were utilized. The 50 3M animals included 11 Tg+ males, 10 Tg+ females, 15 Tg− males, and 14 Tg− females. The 9M group consisted of 11 Tg + males, 11 Tg+ females, 10 Tg − males, and 10 Tg − females. The animals were individually housed with free access to water and Purina rodent chow and were maintained on a 12-h light/dark cycle. All behavioral testing was performed during the light phase of this circadian cycle. Animal weights were recorded at weaning and following the completion of behavioral testing at 3M or 9M.

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2.2. General testing protocol

2.5. Y-maze

Five weeks of behavioral tests were scheduled for each age group so that all animals reached 3M or 9M at the conclusion of their behavioral testing. Animals were administered tests contrived to evaluate both sensorimotor capabilities and cognition in the following sequence: open field task, beam task, string task, Ymaze, water maze acquisition and retention, circular platform task, visible platform, passive avoidance task, and active avoidance task.

To measure spontaneous alternation behavior and exploratory activity, a black Y-maze with arms 21 cm (long) by 4 cm (wide) with 40-cm walls was used. Each animal received one trial, in the course of which the animal was placed into one of the three alleys and allowed free exploration of the maze for five min. Alternations and total number of arm choices were recorded. Spontaneous alternation, expressed as a percentage, refers to ratio of arm choices differing from the previous two choices to the total number of arm entries.

2.2.1. Open field For open field testing of activity and exploratory behavior, an open black box (81 × 81 cm) with 28.5 cm walls was used. The box floor was painted with lines to demarcate 16 squares (20× 20 cm each). For the single trial, each animal was admitted to the center of the enclosure and permitted to explore the interior for 5 min. The total number of line crossings and rearings was recorded. 2.3. Beam task For evaluation of balance and general motor function, a 1.1-cm wide dowel beam was fixed between two support columns, 45.7 cm above a padded surface. At either end of the 50.8-cm long beam was attached a 14 ×10.2 cm platform. Each animal was administered three trials during the single day of testing. The animal was placed in a perpendicular orientation at the center of the beam and released for a trial interval of 60 s. The time required for the animal to fall from the beam was noted for each of the three trials. Alternatively, if the animal remained on the beam for the duration of the trial or escaped to either platform, the maximum interval of 60 s was recorded. The score for each trial, the average of the three trials, and the number of escapes were recorded for each animal.

2.4. String task In order to measure forepaw grip capacity and agility, cotton string was tautly suspended 50.8 cm above a padded surface in the beam apparatus described above. Initially, the animal was permitted to grasp the string only by the forepaws and was then released. In the course of the 60-s trial, a rating system was used to assess each mouse [24]: 0, animal unable to remain on string following release; 1, hangs by two forepaws for 60 s; 2, attempts to climb onto string; 3, two forepaws and one or both hindpaws around string; 4, four paws and tail around string with lateral movement; 5, escape.

2.6. Water maze (submerged platform) A 100-cm circular inflatable pool was used to measure acquisition and memory retention. For purposes of analysis, the pool floor was divided into quadrants: QI, QII, QIII, and QIV. An indiscernible 9-cm platform was positioned in QII, 1.5 cm below the water surface. Testing involved four trials per day over 10 days. In the course of daily testing, the animal was admitted successively into each of the quadrants and allowed to swim for a maximum of 60 s. Upon locating the platform (or after 60 s) the animal was permitted to remain on the platform for 30 s prior to the next trial. Latency to find the platform for each of the four trials and the average of the trials was recorded for each animal. On the day following the 10 days of acquisition testing, memory retention was determined in a single 60-s probe trial. The submerged platform was removed from the water maze and the animal was released into the quadrant opposite that into which the submerged platform had been placed for the acquisition trials. Trials were videotaped for subsequent analysis of swim path and swim speed. The percent of time spent in each quadrant was determined and statistically analyzed.

2.7. Circular platform task An enclosed 69-cm circular platform with 16 holes (4.5 cm) equidistantly spaced 1.3 cm from the periphery was used to measure spatial learning/memory according to our basic methodology [28]. Holes designated as 4, 8, 12, and 16 permitted attachment of an escape box immediately beneath the hole. Visual cues were provided along the interior of the 15-cm enclosure wall and on the interior of a black curtain that isolated the apparatus from its surroundings. Two 150-watt floodlamps mounted 76 cm above the platform and a highspeed fan mounted 15 cm above the platform provided an appropriate degree of aversive stimuli for experimental animals to escape the circular platform. Following 1 day of shaping, mice were tested for 7 days. Each animal received one trial per day in which the animal

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was released at the center of the platform and allowed a maximum of 5 min to locate and enter the escape box. The escape location was moved after each trial but remained constant for any given animal over the 7-day testing interval. For each trial, the total number of choices (‘head pokes’) and latency to find the escape hole were recorded. In addition, a semi-quantitative analysis was devised in order to account for those trials in which the animal failed to enter the escape box. Scale values were assigned according to the number of errors: 1= 0–1 errors, 2= 2 – 4 errors, 3=5 – 9 errors, 4 ] 10 errors, 5=no entry. For semi-quantitative analysis, an average of the 7 days of trials for each animal was used to compare groups.

2.8. Water maze (6isible platform) For measurement of the ability to locate a variably placed visible platform in a 100-cm circular inflatable pool, the 9-cm circular escape platform (used in the memory acquisition task) was elevated and blackened so as to be clearly evident 0.8 cm above the surface of the water. Attached above the platform was a 10× 40 cm black and white foam ensign. Testing extended for 4 days with four trials per day. The animals were admitted to the pool at the same location for all trials, but the platform was moved to a new quadrant for each of the four trials. Animals received a 30-s rest on the platform between trials. Latency to find and ascend the visible platform was measured to a maximum of 60 s.

2.9. Passi6e a6oidance To measure the ability of mice to remember a foot shock delivered 24 h earlier, a two-compartment enclosure with shockgrid floor was used. During the first day of testing, the animal was placed into the smaller lighted compartment. A door to an adjacent darkened compartment was opened and the latency for the animal to enter the darkened region was measured. Upon complete entry into the adjacent chamber, the door was closed and the animal received a 2-s foot shock. The animal was then removed from the chamber and returned to its cage. Following a 24-h interval, the animal was readmitted to the lit chamber, the door again opened and the latency to enter the adjacent darkened compartment was measured (to a maximum of 5 min).

2.10. Acti6e a6oidance Conditioned avoidance responses (CAR) were measured by means of a 22.9 ×20.3 × 44 cm enclosure with a shock grid floor. A 43.2-cm vertical pole and two elevated lateral beams within the enclosure permitted escape from the applied floorshock. During each trial, the animal was exposed to 5 s of bright illumination

which served as the conditioned stimulus (CS). The conditioned stimulus was followed by a 15-s foot shock (US). If the animal exhibited a CAR by escaping the grid floor during the 5-s CS interval, both CS and US were discontinued. All animals received ten trials per day for 12 days. For each animal, the percentage of CARs over the ten daily trials was reported for each day of testing.

2.11. Statistical analyses of beha6ior For the purpose of statistical analysis, animals were grouped by genotype, sex, and age. 3M and 9M animals were independently analyzed by means of analysis of variance (ANOVA) according to genotype and sex. In addition, 3M animals within each of the four sex/genotype groups were compared with 9M animals by ANOVA. For each sensorimotor task, animals whose performance placed them at greater than two standard deviations from the mean were excluded from the statistical analysis for that task. For the open field, beam, water maze retention (submerged platform) and Y-maze tasks, planned comparison analyses of variance between animals of each genotype (Tg+ , Tg − ) and sex were performed independently for both the 3M and 9M groups. Also, 3M animals of each genotype and sex were compared with 9M animals of identical genotype and sex by ANOVA. For the water maze acquisition (submerged platform), water maze visible platform, circular platform (escape latency and number of errors) and active avoidance tasks, planned comparison repeated measures ANOVAs were performed between Tg+and Tg− animals for each sex/genotype group. Analyses of variance were likewise used to compare 3M and 9M animals of the same genotype and sex. For the string task, circular platform semi-quantitative error rating and passive avoidance task, a non-parametric analysis of variance, the Kruskal–Wallis ANOVA, was used. All behavioral measures were further evaluated by means of a comprehensive correlation matrix and factor analysis with Statistica analytical software. For these analyses, the mean of repeated measures trials (water maze acquisition and visible platform tasks, circular platform task, and active avoidance task) over all days of testing was used. For water maze retention, the relative percentage of time spent in QII was utilized. Factor analysis was performed with two–ten measurements for each manifest variable on both raw and standardized data. Factor loadings were determined following varimax and biquartimax rotations.

2.12. Sur6i6al analysis In order to maximize accuracy, survival analysis was performed on all progeny of the male founder. This

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group of 313 animals consisted of 60 Tg+ females, 80 Tg− females, 63 Tg + males, and 110 Tg + males. For all animals, survivorship analysis effectively commenced at weaning (4 – 5 weeks of age). Cox’s F-test and log-rank tests (Statistica) were used to compare groups and a Kaplan – Meier product-limit estimator was used to illustrate group differences.

3. Results Table 1 summarizes all of the reported differences between Tg+ and Tg− mice.

3.1. Sensorimotor tasks 3.1.1. Open field and Y-maze acti6ity At 3M, Tg+ mice exhibited greater open field activity than Tg − control animals [F(1,48) = 6.08, PB 0.02; Fig. 1A]. In particular, 3M Tg+ males were more

149

active than 3M Tg− males, as measured by both the open field task [F(1,24)= 6.48, P B 0.02; Fig. 1A] and total number of arm choices in the Y-maze [F(1,24)= 11.76, PB 0.005; Fig. 2A]. No such differences in activity were evident between 3M Tg+ and 3M Tg− females. There were no differences in either open field or Y-maze activity between Tg+ and Tg− animals at the 9M timepoint. However, 9M Tg+ males exhibited greater Y-maze activity (arm entries) than 9M Tg− males. When all Tg+ males (3M+ 9M) were compared to all Tg− males, the greater open field activity of the Tg + males was highly significant [F(1,43)= 9.90, PB 0.005]. This plenary increase in open field activity by Tg+ males was corroborated in the Ymaze, wherein all Tg+ males exhibited a greater number of arm choices compared to all Tg − males. The aforementioned greater open field and Y-maze activity of all Tg + males was responsible for the significantly greater activity of all Tg+ animals (3M+ 9M) in these tasks compared to all Tg− animals.

Fig. 1. Sensorimotor tasks (mean+ S.E.M.): (A) Activity as measured by open field line crossings. 3M Tg + males \ Tg − males, P B0.02. 3M Tg+ \Tg −, P B0.02. (B) Equilibrium and agility as measured by time balanced on beam. 3M Tg+ B Tg −, P B0.03. (C) Grip strength and agility as measured by forepaw string suspension.

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Table 1 Summary of differences in behavior between Tg+ and Tg− mice by age and gendera Age in months 3M

Open field activity Balance beam task String task Y-maze arm entries Y-maze alternation Water maze acquisition Water maze retention Water maze visible platform Circular platform errors Circular platform latency Circular platform error rating Passive avoidance Active avoidance a

9M

All

Male

* *

*

Female

All

** *

3M+9M Male

Female

*

Progressive impairment

All

Male

Female

* **

** *

*

**

*** *

All Tg−

All Tg+

***

**

Tg+ M

Tg− M

Tg+ F

Tg− F

**

***

*

* *** *** **

* *** *** *

*

*

**

***

** **

**

** * *** *** *

†

† *

†

**

*

*

*

Significant difference at: * = PB0.05; ** = PB0.01; *** = PB0.001. † = no impairment at 3M, but significant impairment at 9M compared to Tg− controls.

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Fig. 2. Y-maze measures (mean +S.E.M.): (A) Arm entries. 3M Tg + males \Tg − males, P B0.01. (B) Percent alternation. 3M Tg + malesB Tg − males, P B 0.05.

3.1.2. Balance beam task In this task, 3M Tg+ mice proved inferior to 3M Tg− animals in agility and balance [F(1,48) = 5.56, P B 0.05; Fig. 1B]. Moreover, for all animals tested (3M +9M), Tg+ mice proved less adept at the beam task than Tg − animals [F(1,90) = 8.0, P B 0.01]. Both male and female animals contributed to this difference by genotype. 3.1.3. String task As a further test of agility and forepaw strength, the string task revealed no differences by genotype at either 3M or 9M (Fig. 1C). However, an age-associated decline in performance of this task was clearly evident for both Tg+ and Tg− animals, as 3M females were more adroit than 9M females [H(1,45) = 18.42, PB 0.001]. This was true for both Tg− females [H(1,24)= 10.35, PB 0.002] and Tg+ females [H(1,21) = 8.31,

PB0.005]. Likewise, 3M males were more adept in the string task than 9M males [H(1,47)= 7.64, P B.001].

3.2. Cogniti6e tasks 3.2.1. Y-maze Spontaneous alternation behavior as measured in the Y-maze did not differ with gender or age (Fig. 2B). However, 3M Tg + males alternated less than Tg − males [F(1,22)= 4.45, PB0.05; Fig. 2B] and Tg + males as a group (3M+9M) alternated less than Tg− males [F(1,43)=4.35, PB0.05]. This decreased alternation of all Tg+ males was primarily responsible for the decreased alternation seen for all Tg+ animals (3M+ 9M) compared to Tg− animals. As noted in the previous section involving measures of sensorimotor ability, total Y-maze arm entries were greater for all Tg+ males (3M+ 9M) than all Tg− males. This gender-re-

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lated difference was responsible for the greater number of Y-maze arm entries shown by all Tg+ animals tested compared to all Tg− animals tested.

3.2.2. Water maze (submerged platform) In the water maze acquisitional testing with a submerged platform (Fig. 3A), there were no differences in overall learning between Tg+ and Tg− animals at either 3M [F(1,34)=0.79, P \0.05] or 9M [F(1,38)= 0.34, P \0.05], nor were there any differences in the rate of learning therein. Moreover, no differences in

learning or rate of learning were present at either time point between Tg+ and Tg− animals of either sex (Fig. 3A) and no age-related differences were evident between all 3M versus all 9M animals [F(1,74)=0.79, P\ 0.05]. Since there were no differences in escape latency between all four sex/genotype groups on the final day (Day 10) of acquisitional testing at both 3M and 9M, all groups at each timepoint had learned the water maze to the same extent and could, therefore, be compared in the memory retention phase of this task. At both 3M and 9M, most groups showed a bias for

Fig. 3. Water maze submerged platform tasks. (A) Acquisition, as measured by latency (mean of four trials per day) to find submerged platform over 10 days. (B) Retention, as measured by quadrant preference during single probe trial without platform after completion of acquisition trials. Among 3M animals, Tg − males and Tg + females failed to exhibit exclusive preference for the acquisition-associated platform quadrant (QII).

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Fig. 4. Circular platform measures during one 5-min trial per day over 7 days of testing. (A) Latency to enter escape box. Among 3M mice, all Tg + \ all Tg −, P B 0.001, and Tg+ females \Tg − females, P B0.001. (B) Errors (‘hole explorations’) prior to entering escape box. 3M Tg + females\ Tg − females, P B0.05.

the former platform-containing quadrant (Q2), indicative of memory retention of the platform’s location (Fig. 3B). One exception, however, was the group of 3M Tg+ females, which failed to show a quadrant preference [F(1,3)= 0.68, P \0.05] and, thus, showed poorer memory retention than 3M Tg− females in this task (Fig. 3B).

3.2.3. Circular platform At 3M, Tg+ males were no different than Tg − males in all measures of circular platform spatial mem-

ory (Fig. 4). In sharp contrast, 3M Tg + females were cognitively impaired compared to Tg− females in all three circular platform measures (Fig. 4): escape latency [F(1,22)= 24.24, PB 0.001], number of errors [F(1,22)= 7.50, PB 0.05], and semi-quantitative error rating (means) [H(1,24)= 12.62, PB 0.001]. This prominent deficit in 3M Tg+ females was primarily responsible for the overall impairment exhibited by all 3M Tg+ animals compared to all 3M Tg− controls (Fig. 4) in escape latency [F(1,47)= 7.61, P B 0.001] and semi-quantitative error rating [H(1,49)= 7.02, P B 0.01].

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Circular platform testing at 9M revealed no differences between Tg+ and Tg− animals in all measures evaluated (Fig. 4). Tg + versus Tg − differences at 3M were not maintained at 9M because of: 1) an age-related decrease in performance of Tg− animals at 9M compared to 3M (latency [F(1,47) =9.68, P B 0.01]), and 2) a stabilization of the impaired performance of Tg+ animals from 3M to 9M. This was certainly the case specifically for Tg+ versus Tg − females at 9M; consequently, no significant differences were evident between these two groups (Fig. 4). However, Tg+ males tended to make more errors than Tg− males at 9M [F(1,19)= 2.95, PB 0.01; Fig. 5]. This trend occurred because of a progressive deterioration in the performance by Tg+ males between 3M and 9M (P= 0.031 for final 6 days of testing; P =0.06 for all 7 days), and not because of any change in performance of Tg− males between 3M and 9M.

3.2.4. Water maze (6isible platform) The visible platform water maze task, in which animals must locate and ascend a highly visible platform, revealed profound differences between Tg+ and Tg − animals (Fig. 5). In this task, the lesser ability of 3M Tg+ mice relative to Tg− controls was not significant [F(1,18)=4.24, P =0.054]. However, at 9M, Tg+ animals were clearly less capable of locating the cued platform compared to Tg− controls [F(1,39) = 14.79, PB 0.001], indicating a progressive impairment between 3M and 9M. This transgene effect at 9M was also present for both males and females, independently (Fig. 5). Indeed, the highly significant genotypic differ-

ences at 9M contributed preponderantly to an overall (3M+ 9M) difference by genotype for all animals tested [F(1,59)= 19.18, P B 0.001]. The poorer overall performance of Tg+ animals in this task is further underscored by the significantly inferior performance of Tg+ males and Tg+ females in comparison to their respective Tg− controls.

3.2.5. Passi6e a6oidance At both 3M and 9M, there were no differences in pre-shock latency to enter the dark chamber among the four genotypic/gender groups (Fig. 6), indicating that all groups at each time point were similarly motivated to enter the dark chamber. For the post-shock trial of memory retention, there was no difference in latencies between Tg+ and Tg − animals at 3M, although 3M Tg− females were less able to remember the previously administered shock than 3M Tg+ females [H(1,24)= 3.85, P= 0.05; Fig. 6]. However, there was no such transgene effect for 3M males, which precluded any overall transgene effect at 3M. At 9M, there was an overall genotype difference [H(1,42)= 4.37, PB0.05] for the post-shock trial, which was not significant for females or males analyzed separately. There also was an overall (3M+9M) transgenic effect for all females tested [H(1,45)= 6.24, PB 0.02]. In both of the latter statistical comparisons, Tg− mice displayed shorter latencies than Tg+ mice. 3.2.6. Acti6e a6oidance There were no differences between Tg+ and Tg− animals in acquisition of active avoidance behavior at

Fig. 5. Water maze visible platform task. Acquisition as measured by latency (mean of four trials per day) to find conspicuously marked platform over 4 days. Among 3M animals, despite a tendency for Tg + animals to present larger latencies than Tg − animals, there were no significant differences by genotype. Among 9M animals, Tg + mice \ Tg− mice, PB0.001; Tg + males \Tg − males, P B0.05; and Tg + females\ Tg− females, P B0.01.

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Fig. 6. Passive avoidance memory retention (mean + S.E.M.) as measured by the ability of mice to remember (24 h later) an electrical shock associated with entry into a darkened chamber. 3M Tg − femalesB Tg+ females, P B0.05; 9M Tg − animals BTg + animals, PB0.05.

either 3M or 9M, nor were there any differences at either age between Tg+ and Tg − males or females (Fig. 7). However, there was an age-related impairment in acquisition exhibited by Tg− males by 9M [F(1,23)= 7.56, PB 0.02]. This contributed substantially to the age-related impairment in acquisition that was evident for all males (Tg+ and Tg− ) tested [F(1,43) =11.20, PB0.002].

3.3. Correlation analysis of beha6ioral measures To further clarify the findings and elucidate inter-relationships between the various sensorimotor/cognitive tasks, correlation analyses were performed using data from all animals in this study. Table 2 indicates the results of this correlation analysis. In measures of animal sensorimotor activity, the most prominent correlation existed between open field activity and Y-maze arm entries (r= 0.642, P B.001). Among cognitive tasks, Y-maze alternation percentage was discordantly correlated with water maze acquisitional latency (r= 0.232, P B 0.05), indicating that superior performance in Y-maze was associated with poor performance in water maze acquisition. However, Y-maze alternation percentage was inversely correlated with both the number of errors (r = − 0.260, P B0.05) and escape latency (r= −0.297, PB 0.01) in the circular platform task, indicating concordance between these two tasks. Water maze task measures were highly correlative with one another. For ‘submerged platform’ water maze testing, a highly significant negative correlation was present between acquisition (latency) and retention (r= −0.626, P B 0.001), while a positive correlation

was evident between water maze acquisition (submerged platform) and performance in the ‘visible platform’ version of the water maze task (r=0.418, PB 0.001). The inverse relationship between acquisition and retention indicates that animals capable of efficiently locating the submerged platform during learning trials spent a greater proportion of time in the ‘platform quadrant’ during the subsequent retention trial. Results from the visible platform version of the water maze and the active avoidance task were also inversely correlated (r= − 0.342, PB 0.01), showing that those animals requiring less time to find the visible platform also demonstrated more conditioned avoidance learning in the active avoidance task. All three measures of circular platform performance (escape latency, number of errors, and semi-quantitative error rating) were highly correlated with each other at PB 0.001 or a higher level of significance.

3.4. Factor analysis of beha6ioral measures Factor analysis of data obtained from the behavioral measurements was performed to effectively extract the underlying relationships among tasks. For simplicity, latent factor names have been derived from primary behavioral task loadings. From all measures recorded for each of the nine behavioral tasks, analysis suggests that six basic latent factors were measured: 1. water maze acquisition and retention measures (WM); 2. circular platform errors (CPE); 3. circular platform latency (CPL); 4. water maze visible platform latency (VPL);

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Task

Sensorimotor OF

Open field activity String task Balance beam task Y-maze alternation Y-maze entries Water maze acquisition Water maze retention Visible platform acquisition Circular platform latency Circular platform errors Circular platform rating Passive avoidance Active avoidance

String

Y-maze Balance

Altern.

Water maze Entries

Acquisition

Circular platform Retention

Visible

Latency

+0.001 −0.05

−0.01 −0.01

+0.001 +0.05

+0.05

−0.01 −0.05 −0.05

Rating

−0.001

−0.05 −0.05 +0.05

−0.05 −0.001

+0.001

+0.001 +0.001

+0.05

+0.05

−0.01

Active

−0.01 −0.05

+0.001

−0.001 +0.001 −0.01 −0.05 −0.001

Passive

+0.05

+0.05

−0.05

Errors

Avoidance

+0.001 +0.001 −0.05

+0.05 −0.01

+0.001

−0.01

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Table 2 Aggregate significant correlations among behavioral tasks

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Fig. 7. Active avoidance acquisition as measured by the percentage of conditioned avoidance responses (CAR) over 12 days.

5. active avoidance CAR learning (AA); 6. activity/exploration (OF,YME) WM accounted for the greatest proportion of the collective variance. CPL and Y-maze spontaneous alternation loaded inversely along a common axis. Only the string task and the passive avoidance task failed to load independently. Thus, factor analysis suggests that each task measured certain components of sensorimotor or cognitive function that were not measured to the same degree by the other tasks.

3.5. Sur6i6al analysis Chi-square comparison of the survival function through 9M between all four groups (Tg+ males, Tg− males, Tg+ females, and Tg− females) revealed a group difference in survivorship (x 2 =11.20, PB 0.02). Further analysis (Cox’s F-test) indicated that the group difference was attributable to genotype [F(38,16)=3.86, P B 0.005] rather than gender [F(32,26)=1.53, P\ 0.05]. Tg + mice had a significantly lower cumulative proportion surviving (higher mortality) than Tg− animals through 9M. This is evident from the Kaplan – Meier product-limit estimator of the survival function (Fig. 8). Expressed strictly in terms of relative mortality, 95.8% of Tg− mice were alive through 9M, but only 84.5% of Tg+ animals reached that timepoint. Nonetheless, all Tg+ and Tg − animals surviving through 9M were in good health.

is the first to thoroughly examine both sensorimotor capabilities and cognitive faculties of APPSW transgenics. Moreover, behavioral measures at 3M and 9M were analyzed not only for age-related transgene effects, but also for sex-related differences. As summarized in Table 2, our results show the presence of sex-related cognitive deficits in APPSW transgenic mice, with several of these deficits being progressive in nature. Because such deficits occur before overt Ab deposition in the brain, it is most likely that elevated brain levels of ‘soluble’ Ab are responsible for the behavioral impairments seen in APPSW mice through 9M. In the circular platform task for spatial learning/ memory, APPSW transgenic (Tg+) mice showed cognitive impairment in all three measures (latency, errors, error rating) at the 3M timepoint. This transgenic impairment was ‘gender-based’ because the higher latencies and greater errors exhibited collectively by 3M Tg+ animals was due to impairment in Tg+ females, but not Tg+ males (Table 2). In contrast to these circular platform impairments in Tg+ animals at 3M, there were no Tg + versus Tg − differences at 9M, because of both an age-related decrease in performance by Tg− animals and no further age-related impairment in Tg+ animals. Importantly, however, a progressive increase in number of errors did occur for

4. Discussion The APPSW transgenic mouse provides a variety of neuropathologies associated with AD, including progressive increases in brain Ab levels and neuritic plaque densities [14,16,31], as well as histopathological evidence for a brain inflammatory response [10] and increased brain oxidative stress [27,30]. However, behavioral changes in APPSW transgenics have not been comprehensively evaluated, particularly at multiple behavioral timepoints. In that context, the present study

Fig. 8. Kaplan – Meier estimator of survival function through 9M. The group difference in survivorship, PB 0.005, is primarily attributable to genotype rather than gender, with Tg + animals exhibiting less survivorship than Tg − animals, PB 0.02.

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Tg+ males between 3M and 9M (Table 2). This progressive effect in Tg+ males further underscores the importance of analyzing behavior in terms of gender, since the effect is masked when the data are analyzed without regard to gender (i.e., males and females combined). It should be mentioned that the circular platform protocol used in this study was adapted by our laboratory from its successful use in rats [1 – 3]. We have previously found that 7 – 8M APPSW transgenic mice are substantially impaired in the circular platform and have suggested that this spatial task offers numerous advantages over more prominently used tasks of cognitive performance in mice [28]. Among these advantages, the circular platform task: 1) does not involve food deprivation, water submersion, or stressful foot shock, 2) has a minimal reliance on sensorimotor skills, and 3) involves three parameters (latency, errors, error rating) that are closely correlated with one another. Moreover, this task appears to be more sensitive than other tasks (i.e., water maze, Y-maze) at detecting age-related cognitive impairment in mice, since 9M animals in this study were collectively impaired in CPL compared to 3M animals. No such age-related impairment was evident for the same animals in water maze or Y-maze performance. Results from the visible platform task were even more noteworthy than those from the circular platform regarding cognitive impairment of Tg+ mice. A progressive impairment in latency to find the visible platform occurred for Tg+ animals between 3M and 9M. Tg+ mice (collectively or by gender) were clearly impaired at 9M, but not at the earlier 3M time point. Researchers have often used the visible platform task in an attempt to discriminate visually-compromised animals, with deficits in this task usually attributed to visual impairment. However, the progressive impairment in visible platform performance presently observed for Tg + animals is not likely to reflect decreased visual acuity of Tg + mice, but rather a true cognitive deficit that correlates well with performance of the same animals in other cognitive tasks (see Table 1). Firstly, if Tg + were visually impaired at 9M, they should have shown impaired acquisition/retention in the submerged platform version of the water maze; however, they were clearly not impaired in that task. Secondly, a number of recent studies are consistent with the visible platform as a cognitive-based task and not as a screening procedure for visual acuity. Lindner et al. [22] found that completely blind rats are not profoundly impaired in locating a visible platform and that normal animals performed poorly in this task following anti-cholinergic treatment with atropine. De Bruin et al. [7] reported that rats with prefrontal cortex lesions performed as well as controls in submerged platform water maze testing, but that lesioned animals were

impaired in subsequent visible platform testing. The authors suggest that lesioned animals had diminished behavioral flexibility in shifting from a ‘spatial’ strategy to a ‘visual’ strategy in order to reach a goal (i.e., lesioned animals failed to inhibit a learned response that had become irrelevant). Additionally, Jackson and Strong [19] found that hippocampectomized rats have a significant ‘orienting’ deficit for recognizing doors in the Lashley III maze and, thus, run past door openings much more than controls. Together, the aforementioned studies strongly support the visible platform task as having a cognitive basis and animals performing poorly in this task as having impaired ‘search’ or ‘recognition’ behavior. This would especially appear to be the case for 9M Tg + animals of the present study, wherein impaired visual acuity is highly unlikely. Interestingly, Hsiao et al. [16] reported impaired visible platform performance of 9–10M Tg+ animals on 2 of 4 test days, but preferred to emphasize the 2 days wherein Tg+ animals were not impaired. In sharp contrast to the clear impairment of Tg+ mice in both circular platform and visible platform tasks, Tg+ mice showed minimal (if any) impairment in Y-maze or water maze (submerged platform) through 9M. Other than a slightly lower Y-maze alternation percentage for Tg+ males at 3M, no other Tg+ effects were present in Y-maze. This was the case irrespective of whether data analysis was performed collectively (males and females combined), or independently evaluated with regard to gender or age. Our results are in contrast to Y-maze data reported in two other APPSW behavioral studies, wherein Tg+ animals showed less spontaneous alternation at 3M [14] or at 10M [16] compared to Tg − controls. Additionally, our results from both acquisition and retention phases of water maze testing did not indicate a substantive impairment in Tg+ animals through 9M when analyzed collectively, by gender, or by age. Our water maze results are contrary to those of Hsiao et al. [16], who reported an impaired acquisition of Tg+ mice at 9– 10M, but not at 3M. Discrepant Y-maze and water maze results between the present study and earlier studies [14,16] could reflect differences in genetic background of Tg+ animals, subtle differences in behavioral testing procedures, and/or different environmental influences. What seems apparent from our data is that the circular platform and the visible platform tasks are more sensitive in detecting cognitive impairment in Tg+ mice than either the Y-maze or water maze (submerged platform). Indeed, both the circular platform and visible platform tasks detected progressive cognitive impairment in Tg+ animals between 3M and 9M (see Table 2). The sensorimotor tasks evaluated in this study exposed several differences between Tg+ and Tg − animals. Although the String task revealed no differences

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in agility and forepaw strength between Tg + and Tg− mice, an age-related decrease in this measure was apparent for both groups. In the balance beam task, Tg+ mice were statistically impaired compared to Tg− animals at 3M, but not at 9M. Since there was no appreciable difference in balance for either Tg+ or Tg− animals between 3M and 9M, it is not surprising that an overall (3M+9M) decrease in balance performance for all Tg+ animals was observed. For both open field and Y-maze activity, Tg+ males showed significantly more activity than Tg− controls at the 3M time point. Indeed, the higher open field activity of all Tg + animals (males and females combined) at 3M was due to higher activity of Tg + males, but not Tg+ females, in comparison to their respective Tg− controls. By contrast, no significant Tg+ versus Tg− differences in open field or Y-maze activity were evident at the 9M timepoint. This was primarily because of an increase in activity shown by Tg− males at 9M compared to those at 3M in both. The higher activity of Tg+ males versus Tg− males at 3M could reflect interactions between increased brain Ab levels present in 2 – 3M Tg + mice [16] and circulating testosterone levels. Alternatively, this higher activity in Tg+ males may reflect interactive developmental effects between testosterone and Ab. Since open field/Y-maze activity involves multiple facets (i.e., general locomotion, exploratory behavior, fear/anxiety), the APP transgene could be affecting one or more of these facets. In any event, our results showing no increase in combined (male + female) Y-maze activity in Tg + mice at 3M are in accord with Holcomb et al. [14], who reported increased activity in doubly mutant APPSW + PS1 transgenics, but not APPSW transgenics, at the same age. No gender-related analysis was reported by Holcomb et al. [14], nor in the only additional study [16] that investigated behavioral effects of APPSW transgenic mice. In the present study, results from active and passive avoidance testing revealed no impairment of Tg+ animals in either task. For example, results from active avoidance testing indicated no differences between Tg+ and Tg − animals at either 3M or 9M, regardless of gender or age. In passive avoidance memory retention, Tg− females were significantly impaired at 3M compared to Tg+ females; indeed, the continued poorer performance of this group at 9M was primarily responsible for a genotypic difference present at 9M. The lack of impairment by Tg+ animals in active and passive avoidance testing is consistent with results reported by Malherbe et al. [23] in PD-APP transgenics, wherein no deficits in active or passive avoidance appear to have occurred at 12M (no actual data was presented in that study). Our results suggest that avoidance tasks may not be sensitive enough to discriminate cognitive impairment in APPSW transgenic mice, al-

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though behavioral testing at time points beyond 9M could reveal impaired performance of Tg+ animals in one or both of these tasks. This is the first behavioral study in any APP transgenic mouse model to involve a correlation analysis between tasks (see Table 1). As such, significant correlations between behavioral measures were observed to fall into three groups: 1) ‘internal’ correlations attributable to task similarity or multiple measures in a single task, 2) correlations between dissimilar tasks that are nevertheless consistent with prevailing theory and prior studies, and 3) correlations without immediately apparent theoretical justification. The three highly significant circular platform measures exemplify internal consistency among the correlations. Similarly, most of the water maze measures were significantly correlated. Correlations of the second type (i.e., those that would appear to comport with the theoretical presuppositions underlying design of the tasks), include the significant association between water maze acquisition and Ymaze acquisition, as well as the highly significant correlation between open field activity and Y-maze arm entries. The more problematic significant correlations of the third type may include those between CPE and open field, balance beam, Y-maze alternations, and Y-maze arm entries. Factor analysis, undertaken to clarify the relationship between the behavioral tasks, suggests that CPE and both Y-maze measures include a substantive ‘exploratory’ component. Additionally, CPE (rather than CPL) more faithfully captures the animal’s predilection toward spontaneous mobility, conventionally measured by the open field task. The AA task was significantly (and inversely) correlated with both water maze acquisition and visible platform tasks. The correlation between AA and water maze acquisition may result from a shared requirement for reference memory, while the correlation between AA and visible platform could reflect the need for associative/recognition processing in both of those tasks. As indicated earlier, the visible platform task is not ideally suited for merely eliminating visually impaired animals; rather, our factor analysis strongly suggests that this task has clear cognitive requirements. Thus, animals must recognize the significance of the visible platform and form the appropriate association for escape. Parenthetically, it should be mentioned that our factor analysis revealed each task of this study to measure certain components of behavior that were not measured by the other tasks. Such measurement of multiple behavioral components through a comprehensive battery of tasks is highly desirable for discerning the true behavioral profile of genetically-manipulated animals. An important issue that should be addressed is the extent to which the APPSW mutation is responsible for behavioral impairments exhibited by APPSW transgenic mice in the present study. Certainly, the most likely

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explanation for impaired behavior shown by APPSW transgenics is an overexpression of mutant APP in the brain, with consequent increases in brain Ab levels. Indeed, given reports of both overexpression of APP695 and elevated brain Ab levels by 2 – 4M in APPSW transgenics [16], this may be the most likely explanation for their impaired behavior in the current study. Nonetheless, it should be noted that APP overexpression and/or elevated Ab levels during brain development (which occurs for several months post-natally) offer the related, but mechanistically different, explanation of neurodevelopmental effects of the APP transgene. Behavioral impairments in Tg+ animals could also be due to ‘compensatory’ processes by other genes in response to the overexpressed APP gene. Additionally, at least some behavioral impairment in APPSW transgenics could be caused by ‘background’ genes immediately adjacent to the APP gene, which are unavoidably inserted into the genome [12]. These background genes, which may themselves be ‘mutated’ [21], have the potential to affect neurodevelopment and/or adult behavior. Yet another possibility is that the insertion of multiple copies of any gene construct could induce behavioral impairment. For example, small amounts of extra genomic DNA in humans with unbalanced translocation karyotypes results in mental retardation. Indeed, such increased genomic DNA could be responsible for the higher mortality rate exhibited by Tg + animals in the present study and in previous APP transgenic studies [16], although extent of APP overexpression in the various transgenic lines may also be a factor [15]. It is important to consider that the cognitive impairment presently demonstrated in Tg+ mice was evident at 3M or 9M—prior to overt Ab deposition and neuritic plaque formation in their brains. Although brain levels of ‘soluble’ Ab are elevated by 2 – 4M in APPSW transgenics [16] and increase further by 6 – 8M [14], we have found no immunostaining for Ab deposition in brains of APPSW transgenics through 8M (unpublished observations), nor have others [14]. Indeed, Ab-containing neuritic plaques are not evident in APPSW transgenics until 10–18M [4,10,16]. Since cognitive impairment in the current study was present before neuritic plaque formation/Ab deposition, we postulate that overt Ab deposition is not necessary for cognitive impairment in APPSW mice. Rather, it is more likely that elevated brain levels of soluble/fibrillar Ab in APPSW transgenic mice are inducing as yet uncharacterized pathophysiologic changes that result in cognitive impairment. These postulates are supported by previous APP transgenic studies wherein cognitive impairments were seen prior to, or without, Ab deposition [9,14,32]. The clinical literature also supports this premise in that: 1) Ab deposition in AD is not correlated with cognitive impairment, and 2) extensive neuritic plaque formation/

Ab deposition has been observed post mortem in aged individuals with no cognitive impairment [8]. Several final points appear warranted regarding our findings in this study. Firstly, the cognitive impairments observed at either 3M or 9M were usually gender-dependent (see Table 1). Therefore, analysis of behavioral data without regard to sex would have masked these deficits in Tg+ animals and/or reflected a deficit present exclusively in Tg+ males or Tg+ females (neither of which the investigator would be aware of without a gender-specific analysis). Secondly, a poorer performance of Tg− animals during aging can negate earlier cognitive impairments exhibited by Tg+ animals. Therefore, Tg+ animals sometimes exhibited cognitive impairment in a given task at the 3M timepoint compared to Tg− animals (i.e., accelerated impairment), but at 9M, no significant difference was present because a temporally normal impairment of Tg− animals became manifest by then. Finally, it should be noted that all Tg+ animals in this study were F1 generation hybrids generated from the same heterozygous progenitor male. As such, their genetic background was reasonably homologous. Using F2 generation hybrids or combinations of generations therein would have eliminated this genetic homology, resulting in increased behavioral variability across individuals because of independent assortment and new combinations of segregating background genes [5]. Particularly since behavioral studies involving transgenic mice are still in their infancy, we reasoned that it is preferable to have less variability in APP expression/Ab levels among Tg+ animals than to decrease the possibility of background gene effects through cross-breeding of multiple generations. In summary, the present study comprehensively evaluated sensorimotor skills and cognitive abilities in APPSW mice through 9M. Our results indicate the presence of cognitive deficits that are progressive in nature for several tasks and often gender-dependent. Moreover, these cognitive impairments are present prior to overt Ab deposition in the brain, suggesting a causative, pathophysiologic role for elevated levels of ‘soluble’ Ab in such relatively early cognitive impairments. It will be important to determine if progressive cognitive impairments continue to be seen in APPSW transgenics at later timepoints, when brain Ab deposition and neuritic plaque formation are prevalent.

Acknowledgements This research was supported by the USF Alzheimer’s and Parkinson’s Disease Research Fund (G.W.A), the Roskamp Laboratories, USF Department of Psychiatry (M.J.M), and the generosity of Robert and Diane Roskamp. We are especially grateful to Patrick Pompl

D.L. King et al. / Beha6ioural Brain Research 103 (1999) 145–162

and Ray Martinez (Department of Biology) who were instrumental in the design of behavioral apparatuses used in this study.

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