Loss of Cholinergic Phenotype in Basal Forebrain Coincides with Cognitive Decline in a Mouse Model of Down's Syndrome

Experimental Neurology 161, 647–663 (2000) doi:10.1006/exnr.1999.7289, available online at http://www.idealibrary.com on

Loss of Cholinergic Phenotype in Basal Forebrain Coincides with Cognitive Decline in a Mouse Model of Down’s Syndrome Ann-Charlotte E. Granholm,*,†,‡ Linda A. Sanders,*,† and Linda S. Crnic§,¶ *Department of Basic Science, †Department of Pharmacology, §Department of Pediatrics, ¶Department of Psychiatry, and the ‡Neuroscience Training Program, University of Colorado Health Sciences Center, Denver, Colorado 80262 Received May 4, 1999; accepted September 30, 1999

Mice with segmental trisomy of chromosome 16 (Ts65Dn) have been used as a model for Down’s syndrome. These mice are born with a normal density of basal forebrain cholinergic neurons but, like patients with Down’s syndrome, undergo a significant deterioration of these neurons later in life. The time course for this degeneration of cholinergic neurons has not been studied, nor is it known if it correlates with the progressive memory and learning deficits described. Ts65Dn mice that were 4, 6, 8, and 10 months old were sacrificed for evaluation of basal forebrain morphology. Separate groups of mice were tested on visual or spatial discrimination learning and reversal. We found no alterations in cholinergic markers in 4-month-old Ts65Dn mice, but thereafter a progressive decline in density of cholinergic neurons, as well as significant shrinkage of cell body size, was seen. A parallel loss of staining for the high-affinity nerve growth factor receptor, trkA, was observed at all time points, suggesting a biological mechanism for the cell loss involving this growth factor. Other than transient difficulty in learning the task requirements, there was no impairment of trisomic mice on visual discrimination learning and reversal, whereas spatial learning and reversal showed significant deficits, particularly in the mice over 6 months of age. Thus, the loss of ChAT-immunoreactive neurons in the basal forebrain was coupled with simultaneous deficits in behavioral flexibility on a spatial task occurring for the first time around 6 months of age. These findings suggest that the loss of cholinergic function and the simultaneous decrease in trkA immunoreactivity in basal forebrain may directly correlate with cognitive impairment in the Ts65Dn mouse. r 2000 Academic Press

Key Words: basal forebrain; acetylcholine; memory and learning; neurodegeneration; nerve growth factor receptors.

INTRODUCTION

Down’s syndrome (DS) is the most prevalent cause of mental retardation and is caused by the presence of all

or part of an extra copy of the 21st chromosome (see 1 for review). The mechanisms by which extra copies of normal genes produce the cognitive phenotype (language and memory deficits, as well as early onset dementia) are not known. However, the animal models used to study these processes suggest that there are specific determinants on the human chromosome 21 that alter brain development in DS (61, 64). The initial approach to an animal model of DS came when the phenotype of mice trisomic for chromosome 16 was recognized as being similar to DS (53). The Ts16 mouse, while providing useful insights into early progression of mental retardation, cannot be used to study the development of dementia, since these mice die at birth. An additional pitfall with this mouse model is that there are genes on chromosome 16 of the mouse that are not present on human chromosome 21, complicating the interpretation of the degenerative alterations seen in the brain of the Ts16 mouse as it relates to DS. Recently, mice with segmental trisomy of chromosome 16 (Ts65Dn mice, see 13, 14) have been used as a model for DS and Alzheimer’s disease (AD). The segment of mouse chromosome 16 that was triplicated in Ts65Dn mice includes material from the long arm of the chromosome just proximal to the gene for the amyloid precursor protein (App) extending to the gene for myxovirus resistance (Mx, see 54). This region corresponds closely to 12–15 mB of the long arm of human chromosome 21 that is present in three copies in the patient with DS (13). These mice have a distinctive phenotype similar to DS (reviewed in 12 and 15) and are healthy enough that extensive cognitive analysis is possible. They have been found to exhibit deficits in both the visible and the hidden platform tasks of the Morris water maze (22, 54), as well as developmental delay in most sensorimotor behavior and reflex systems (34, 35). Individuals with DS exhibit a normal septohippocampal cholinergic system at birth (38) but this system degenerates in early adulthood, similar to the degeneration seen in patients with AD (5, 40, 70). Almost all patients with DS have developed AD by their fourth decade (see e.g., 10). The mechanism for this spontane-

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ous degeneration of cholinergic neurons in the DS patient is not known, and since similar atrophy of cholinergic basal forebrain neurons occurs with age in the Ts65Dn mouse (35), valuable mechanistic data may be gleaned from this mouse model of the disease. Ts65Dn mice may also be valuable in terms of developing potential therapeutic intervention for patients with both DS and AD. Most animal models for degenerative diseases have previously relied on aged animals that exhibit a partial loss of cholinergic neurons or lesion models in young adult animals (9, 11, 18), none of which closely mimic the gradual and transmitter-specific loss of neurons that occurs in the patient. Therefore, the Ts65Dn mouse is an interesting and valuable model system that complements existing model systems using transgenic or excitotoxic approaches to programmed cell death in the CNS. One potential agent that has been studied extensively in degeneration of cholinergic forebrain neurons is nerve growth factor (NGF; 19, 40 for review). It has been well documented that cholinergic forebrain neurons are dependent on NGF for their development and maintenance of phenotype (6, 7, 21, 25, 28). This neurotrophic factor is synthesized in the target regions, cerebral cortex and hippocampus (28, 30), and is then transported retrogradely to the cell bodies in the forebrain (20, 59). Administration of NGF to adult rodents or nonhuman primates with lesions of the cholinergic pathways can rescue axotomized cholinergic neurons (6, 21, 39, 65, 67). These findings suggest that NGF may be a therapeutic tool for patients with AD. There are also postmortem findings from AD brains that suggest a deficient NGF system in the patient. Many investigators have shown that the high-affinity receptor, trkA, as well as trkA mRNA, is significantly decreased in neurons of the basal forebrain and cerebral cortex of patients with AD (2, 3, 32, 46, 47, 56), and they have also found that immunoreactivity to the NGF protein itself is decreased in basal forebrain of these patients (45). Since the levels of NGF are not altered (37, 48), and in some studies have even been found to be elevated in the hippocampus and cortex of AD patients (23, 49, 58), this suggests a specific deficiency either in NGF release from postsynaptic neurons, in uptake of the protein into the terminals, or in transport to the cholinergic cell body in the forebrain. These alterations in the NGF pathway finally lead to significant decreases in NGF protein in the cholinergic basal forebrain cell bodies (45) and subsequent loss of cholinergic phenotype. Interestingly, animal models of AD, such as the aged rat or nonhuman primate, have shown that exogenous NGF administration can enhance the cholinergic systems, alleviate age-related cognitive impairment, and upregulate both high- and low-affinity NGF receptors, despite the fact that NGF levels were unaltered or elevated in the target region to begin with (4,

25, 26, 41, 42, 57). A similar finding was obtained from studies of the Ts16 trisomic mouse, where age-related degeneration of cultured or transplanted cholinergic neurons was prevented with continuous administration of NGF (8, 33). Therefore, we investigated the density of the high-affinity NGF receptor in the present study, in order to compare a potential loss of this receptor with the timing of cholinergic degeneration. Since a careful stereological analysis of the extent of cholinergic cell loss in 6-month-old Ts65Dn mice had already been performed (see 35), the overall purpose of the present study was not to evaluate the total number of neurons in cholinergic forebrain nuclei, but rather to determine the time frame during adulthood at which the cholinergic degeneration is initiated in the Ts65Dn mouse. In addition, the study aimed to evaluate a possible biological mechanism for this cholinergic degeneration, by examining the density and pattern of NGF receptor expression in this brain region during the loss of cholinergic neurons. Finally, the functional consequences of the neuron loss were explored by examining behaviors dependent upon basal forebrain cholinergic neurons, reversal of spatial discrimination learning in a T-maze. MATERIALS AND METHODS

Animals Mice with partial trisomy of chromosome 16 were produced by M. Davisson at Jackson laboratories according to the following protocol. Testes of DBA/2J mice were irradiated twice with 600R from a cesium-137 source. Thereafter, irradiated males were bred with C57Bl/6Jeicher females, and F1 progeny were scored for chromosome (Chr) 16 reciprocal translocations that produced a small translocation chromosome consisting of the centromere of mouse Chr 17 attached to a segment of mouse Chr 16. Mice with translocations were bred for 19 generations (3 years) to get a spontaneous nondysjunction event that led to mice with two copies of the normal Chr 16 plus the small translocation chromosome. The trisomy is maintained by mating female carriers of the partial trisomy (males are sterile) to C57Bl/6Jeicher 3 C3H/HeSnJ F1 males. For the present studies, male mice were bred in our laboratory from stock obtained from The Jackson Laboratories. They were genotyped by FISH using a probe for the centromere of mouse Chr 17. Animals with retinal degeneration due to homozygosity for the mutation carried by C3H mice were screened by amplifying the rd gene followed by restriction digest to detect the mutation (52) and discarded. Mice were maintained on a 12/12-h light/dark cycle (light onset 0700 h), and free access to tap water and Agway mouse chow and were group housed until testing. They were tested at 5–7 h after light onset. The morphological phenotype of the trisomy is extremely subtle; thus, in addition to the

CHOLINERGIC PHENOTYPE LOSS IN A MURINE DOWN’S SYNDROME MODEL

morphological analysis, all behavioral testing could be conducted blind to the genotype of the animals. For color discrimination and reversal, 22 mice averaging 9 months of age (range 8–11 months) were used. These animals had had auditory startle habituation testing and laterality assessment before discrimination training. For spatial discrimination and reversal training, 27 mice of two age groups averaging 4.7 and 8.1 months were used, as age was a variable of interest in this study. These mice were tested for startle habituation at weaning. Two of these mice provided subjects for the morphological analysis, while 3 additional animals of each group at each age (4, 6, 8, and 10 months) were used for the morphometric studies. These mice had been tested for long-term habituation of the auditory startle response at 39 days of age but were otherwise group housed with same sex littermates until sacrifice. Behavioral Testing Black/white discrimination and reversal. Mice were tested for side preference and extent of laterality by determining the arms entered in 10 sequential exposures to a dry T-shaped maze. They were then trained to escape to the white arm of a T-shaped maze immersed in a tub of water kept at 19°C, as described by Denenberg et al. (16). One arm of the maze was painted white and the other black and the maze could be inverted so that arm color had a random relationship to position (left or right side), by using a carefully designed sequence (27). A ladder of hardware cloth allowed the mice to exit the water when they had reached the end of the correct arm and the arms of the T curved back upon the stem so that the mice could not see the escape ladder when they made their choice. Mice were dried with a towel and kept under a heat lamp until the start of the next trial and were run in squads of five to six, so that the intertrial interval was never less than 5 min. Mice were placed in the stem of the maze and allowed to swim down an arm. Mice making incorrect choices were allowed to swim to the opposite arm to escape and those that did not reach the ladder within 1 min were guided to it. The time required to reach the escape ladder and the number and sequence of arm entries were recorded. Ten trials were run each day until a criterion of 8/10 correct trials for 2 consecutive days was reached, after which contingencies were reversed, so that mice had to escape from the black arm and run to the same criterion as on the initial task. Two further reversals of contingency were carried out. Spatial learning and reversal. Left/right discrimination was taught to a separate group of animals using a similar T-shaped maze except that all walls of the maze were gray and the mice were each trained to escape to their nonpreferred side, as determined by the laterality testing described above. Reversal training for this group consisted of requiring the mice to shift to oppo-

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site side of the maze to escape and only one reversal of contingencies was performed. Laterality variables were analyzed together by multivariate analysis of variance. Learning and reversal variables were analyzed by repeated measures analysis of variance. Immunohistochemistry and image analysis. Male mice were anesthetized with Avertin and sacrificed at 4, 6, 8, and 10 months of age by transcardial perfusion with 4% paraformaldehyde with 2% picric acid. The brains were dissected and postfixed for 48 h in the same fixative, after which they were transferred to 30% sucrose in phosphate buffer. Tissue sections of forebrain were processed for immunohistochemistry according to our previous protocols (4). Briefly, free-floating sections (30 µm thick) were rinsed in 0.1 M PBS with 0.05% Triton X-100 and treated with 0.3% H2O2 to inhibit residual endogenous peroxidase. Background staining was blocked by a 1-h preincubation with 3% normal goat serum and 2% bovine serum albumin in 0.1 M PBS. Sections were then incubated for 48 h with antibodies directed against the acetylcholine synthetic enzyme choline acetyltransferase (ChAT, Chemicon, 1:1000) and the high-affinity nerve growth factor receptor trkA (generously provided by Dr. L. Reichardt). Specificity of the trkA antibodies versus trkB and trkC receptors has been verified previously (62).Thereafter, sections were incubated for 1 h at room temperature with biotinylated anti-rabbit IgG as a secondary antibody. Finally, sections were incubated with the ABC Elite substrate (Vector Laboratories Inc., Burlingame, CA) and developed with a nickel ammonium-intensified diaminobenzidine (DAB) reaction (8 min, 20 ml 0.1 M PBS, 0.01g DAB, 100 ml 8% nickel ammonium sulfate, and 68 ml 3% H2O2). Controls included sections incubated in the absence of primary or secondary antibody as well as preincubation with the appropriate antigen. Three washes of 0.05% Triton X-100 in 0.1 M PBS were carried out between each step of the staining procedure. In order to control for intergroup staining variability, all steps of the immunohistochemical staining, including the DAB reaction, were performed in the same solutions and times for all groups using tissue wells with plastic mesh bottoms on an orbital shaker table. Sections were mounted on slides, air-dried, dehydrated in increasing concentrations of ethanol followed by xylene, and coverslipped with DTX mounting medium. They were studied in a Nikon Eclipse 600 microscope. Every 10th section throughout the forebrain region was mounted on glass slides and processed for routine histology using cresyl violet staining. Image analysis of cell body staining and cell sizes in the forebrain cholinergic nuclei was performed using the NIH Image analysis program, on every 10th section of both trkA- and ChAT-immunostained sections throughout the medial septal nucleus (MSN) and ventral diagonal band (VDB) nuclei. The image analysis

FIG. 1. (A) Visual discrimination learning. Data are the latency to reach the escape ladder in the water maze on the first day of spatial discrimination learning and three subsequent reversals of that discrimination. While the first day of each discrimination and reversal is shown, the Ts65Dn mice were significantly impaired compared to control mice on the first 2 days of testing (P 5 0.008). All mice were over 6 months of age at the time of testing. (B) Spatial discrimination learning and reversal. Data are the latency to reach the escape ladder in the water maze on the first day of spatial discrimination learning (left columns in the figure) and the first day of reversal of contingencies on that

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measurements were performed blindly by two independent investigators from which means were then established. Image is written using Think Pascal from Symantec Corporation, and the complete source code is freely available. Image can be used to measure area, average gray value, and path lengths and angles of cellular components. Spatial calibration is supported to provide real world area and length measurements. Density calibration was performed against an optical density calibration curve that takes into account and subtracts the background from each section that is measured. The gray scale value is within the range of 0 to 256, where 0 represents white. The first (most rostral) section of MSN was selected randomly for each animal, using the complete medial fusion of the corpus callosum as the most rostral landmark for both MSN and VDB. Thereafter, every 10th section was used in the image analysis measurements, until the medial fusion of the anterior commissure, which provided the caudal restriction of the measured field. Since the sections were 30 µm in thickness, this rendered a sample distance of more than 270 µm to ensure that no neuron was counted twice. To further ensure that treatments were equal between the two groups, sections from all groups were processed together simultaneously and in the same baths using plastic mesh incubation wells during the entire immunohistochemical process. Background staining density measurements revealed that background staining was within 5 units from each other in each group, further supporting equal treatment during staining. The mediolateral and the dorsoventral area of the MSN and the VDB were first measured in a group of trisomic animals (4 and 8 months old) and an age-matched group of controls, in order to determine whether the Imagegenerated cell packing density measurements were dependent on differences in total volume of the nucleus in the different groups (Figure 5A). The mean staining density, packing density, and cell size values were generated from .200 neurons in each animal (counting all the neurons in each section in three sections for each area). Only cells that exhibited a visible nucleus and at least two processes were characterized as neurons. For further details on the image analysis technique, see Ref. (4). RESULTS Behavioral Results

Black/White Discrimination Learning and Reversal Consistent with prior findings with the trisomic mice, they were significantly smaller than their litter-

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mate controls (28.0 vs 34.6 g, Student’s t 5 3/31, df 5 19, p 5 0.004). One of the 22 mice trained on this task (a Ts65Dn) did not learn the initial discrimination and was omitted from the analysis. There was no difference between trisomic and control mice in the tendency to spontaneously alternate between arms of the dry Tmaze, nor in the direction nor extent of their side preference. The trisomic mice were more likely to have to be forced to the correct arm on the first five trials of initial learning of the discrimination (7/10 Ts65Dn vs 2/11 controls, p 5 0.025 by Fisher’s exact probability test); that is, they had not found the correct arm within 60 s. This slowness to learn the logistics of the task was also revealed in increased latencies to reach the correct arm in the first 2 days of discrimination learning for the segmentally trisomic animals (F 5 8.79, df 5 1,19, p 5 0.008 by multivariate analysis of variance, Fig. 1A). No other variables differed significantly between groups during initial learning and three reversals of contingency. Thus, aside from initial slowness in learning how to escape the maze, visual discrimination learning and reversal did not differ between Ts65Dn mice and their littermate controls. Spatial Discrimination Learning and Reversal For this group of animals, who were not significantly different in body weight by age or genotype, there was a significant effect of genotype on the extent of lateralization, with the trisomic mice being more lateralized than the control mice (64 vs 56% of responses to preferred side, p 5 0.05). To determine whether loss of cholinergic neurons described below had functional consequences for this behavior, the animals were split into those less than (average 4.7 months) and those older than (average of 8.1 months) 6 months of age. Both age and segmental trisomy status had a deleterious effect on spatial learning and reversal, with the older trisomic mice most affected. All mice, however, did learn both discriminations, the differences between groups being apparent in the initial half of learning. For simplicity, the differences on the first day of learning are presented. Spatial discrimination. For initial discrimination learning, there was no difference between groups in the number of correct responses; however, both the number of arms entered (a measure of errors) and the latency to enter the correct arm showed significant effects of age and genotype. Segmentally trisomic animals entered more arms than control mice ( p 5 0.028) and took longer to get to the correct arm ( p 5 0.013, Fig. 1B). Older animals showed the same increase in arm entries

task (right columns in the figure). During discrimination learning, the two segmentally trisomic groups together showed a trend toward being slower to reach the goal than control mice (P 5 0.086). During reversal learning, both carrying the segmental trisomy (P 5 0.038) and being over 6 months of age led to increased trial latency (P 5 0.044).

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TABLE 1

Immunohistochemical Detection of Cholinergic Markers

Performance on the First Day of Spatial Reversal Training

Antibodies directed against the synthetic enzyme for acetylcholine, ChAT, were used to label cholinergic forebrain neurons. Morphological evaluation revealed that there was a progressive loss of ChAT-immunoreactive neurons in Ts65Dn mice, starting at 6 months of age and continuing throughout the experiment (10 months of age, see Fig. 3). ChAT-immunoreactive neurons in the 4-month-old group appeared to be similar in both size and morphological appearance in both groups, while there were significant differences between the groups at the later time points (see Fig. 3). The forebrain neurons appeared shrunken, with sometimes pyknotic nuclei and scattered debris in the Ts65Dn groups at 6, 8, and 10 months of age, as can be seen in Fig. 3. Image analysis (see Materials and Methods above) of sections stained with ChAT antibodies revealed that the MSN and VDB exhibited a significant shrinkage of ChAT-immunoreactive neurons. The average cell size of the different groups is shown in Figs. 4A and 4C. The maximal decrease in cell size in the MSN had occurred at 8 months of age, where the neurons in Ts65Dn mice were only 64% of the control neurons in terms of average cell body size (Fig. 4A). There was also a trend toward a decrease in staining density for ChAT within each neuron in the 6- and 8-month groups (see Figs. 4B and 4D), even though the differences between the two groups did not reach statistical significance. A significant variability in staining intensity was seen with age in both groups, as shown in Fig. 3. ChATimmunoreactive neurons could be found in both groups from approximately the level of 11.2 to 10.3 from Bregma for the MSN region and from 11.5 to 10.4 from Bregma in the VDB region. The landmarks used for MSN were the fusion of the corpus callosum (rostral), the islands of Calleja (lateral), and the fusion of the anterior commissure (caudal). The landmarks used for VDB were the fusion of the corpus callosum (rostral), the ventral pallidum (lateral and inferior), and the medial forebrain bundle (caudal). There was no difference between Ts65Dn mice and controls in terms of the rostrocaudal extension of ChAT-immunoreactive neurons. Area measurements of the nuclei were also performed in the dorsoventral and the mediolateral aspects. As can be seen in Fig. 5A, there was no difference in the measurements of the nuclei in these aspects either, concluding that the volume of the MSN and the VDB nuclei did not appear to be altered in the Ts65Dn mice compared to controls, at least not between agematched groups up to 8 months of age. However, image analysis revealed a significant decrease in the packing density of cholinergic neurons (expressed as percentage of change from controls) between the two groups, both in the MSN and in the VDB (see Fig. 5B). Mean packing density of ChAT-immunoreactive neurons for trisomic mice was 69 6 7 (percentage of control; n 5 9)

Group

Arms entered

Number correct

Ts65Dn ,6 months Control ,6 months Ts65Dn .6 months Ts65Dn .6 months Ts65Dn Control Younger than 6 months Older than 6 months

22.5 6 2.6 17.7 6 5.5 31.1 6 11.6 22.5 6 6.1 27.2 6 9.5* a 20.4 6 6.1 20.1 6 4.8* b 26.5 6 9.8

3.7 6 2.0 6.3 6 1.5 3.0 6 1.9 5.1 6 2.1 3.3 6 1.9** a 5.6 6 1.9 5.0 6 2.2 4.1 6 2.2

Note. Values are the means and standard deviations for each group. * P , 0.05 and ** P , 0.005. a Compared to Control group. b Compared to animals older than 6 months.

( p 5 0.012) while the increase in latency did not reach significance ( p 5 0.085). The older animals required more trials to reach the criterion for having learned the task (44 vs 30 trials, p 5 0.021). It is possible that the slight but statistically significant difference (8%, see previous paragraph) in the strength of side preference between the genotypes contributed to the difficulty in learning of the task by the segmentally trisomic mice. Spatial reversal. For reversal learning, genotype significantly affected the number correct on the initial day of testing with segmentally trisomic animals executing fewer correct responses than controls ( p 5 0.004, Table 1). Age grouping significantly affected arms entered ( p 5 0.022, Table 1) with the older animals entering more arms on each trial. Latencies to complete the trial were negatively affected by both older age ( p 5 0.038) and segmental trisomy ( p 5 0.044) on the first day of testing (Fig. 1B). As with initial learning, the reversal effects were all in the direction of poorer performance for older and segmentally trisomic animals. On all variables, the days effect was significant, indicating that all animals learned the task, so that the differences between groups all occurred in the initial stages of learning. Routine Histological Staining

Sections from the basal forebrain of mice that were 4, 6, 8, and 10 months of age were stained with cresyl violet to determine the gross morphological appearance and volume of this area. As can be seen in Fig. 2, there were no observable alterations in the basal forebrain of Ts65Dn mice, even at 10 months of age (Fig. 2e). The number and density of neurons in this region appeared to be similar in age-matched trisomic and control mice, upon gross morphological examination. There was no observable alteration in other elements in the basal forebrain, such as vascularization or glial cell density, between the two groups.

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FIG. 2. Sections from Ts65Dn mice (Ts, left column; a, c, e) and age-matched controls (C, right column; b, d, f) stained for routine histology, using a cresyl violet staining protocol. The ages shown in this figure are 4 months (a, b), 6 months (c, d), and 10 months (e, f). The age for each row of figures is also indicated as numbers in the lower left corner. As can be seen in this figure, no gross differences could be observed between the trisomics and controls at any age studied in terms of overall neuronal density within the basal forebrain. There were no alterations that could be detected in other elements, such as glial or vascular cells, between the groups at any age examined, and no gross degenerative processes were observed. Bar in f, 125 µm.

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FIG. 4. Bar graphs illustrating the image analysis results for ChAT immunoreactivity in terms of mean cell size (A, MSN; C, VDB) and average staining intensity within the neurons (B, MSN; D, VDB). White bars, controls; solid black bars, Ts65Dn mice (n 5 3 per time point and group). Note that cell body size (expressed as mean square micrometers per animal) was similar between the two groups at 4 months (A, C), but then gradually decreased in the trisomic group (black bars), compared to controls. Statistical analysis with ANOVA revealed a statistical difference between Ts65Dn mice and controls in terms of cell size at 8 months (P ,0.01) and 10 months (P , 0.001) of age in the MSN and at 8 months of age (P , 0.05) in the VDB, as well. As can be seen in B and D, there were no differences in terms of average staining intensity (expressed as the mean measurement of arbitrary units of 0–256 with background subtracted, using an automated image analysis system) for ChAT antibodies within either MSN (B) or VDB (D) between the two groups. All mean values are expressed with SEM and statistics were performed using an ANOVA with Tukey–Kramer post hoc test.

in the MSN and 52 6 6 (percentage of control; n 5 9) in the VDB (deviations are SEM throughout the study). For controls these values were 100 6 9 (n 5 9) in the MSN and 100 6 6 (n 5 9) in the VDB. Thus, a loss of 30% of ChAT-immunoreactive neurons was seen in the MSN, and a loss of 48% of the ChAT-immunoreactive neurons was seen in the VDB of trisomic mice, compared to age-matched controls. Statistical analysis, using a two-way ANOVA, revealed significance between

the two groups, both in the MSN (P , 0.05) and in the VDB (Fig. 5B; P , 0.001). Immunohistochemical Detection of the High-Affinity NGF Receptor, trkA

The alterations described for ChAT-immunoreactive neurons in trisomic mice above were paralleled with similar alterations in the immunoreactivity for the

FIG. 3. Sections from basal forebrain incubated with ChAT antibodies. The area shown throughout this figure is the medial septal nucleus, MSN. The microphotographs on the left are sections from Ts5Dn mice (Ts; a, c, e, and g), while the right side of the figure shows sections from controls (C; b, d, f, and h). The ages depicted are 4 months (a, b), 6 months (c, d), 8 months (e, f), and 10 months (g, h) and each row is labeled with the age in months (lower left corner). Note the progressive loss in ChAT immunoreactive neurons in the Ts65Dn mice (Ts, left side of panel), compared to controls (C). Shrinkage of ChAT-immunoreactive neurons can also be seen in MSN sections from Ts65Dn mice. Bar in h, 75 µm.

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FIG. 5. Bar graphs depicting (A) the mean areas of MSN and VDB in trisomic animals (black bars) and age-matched controls (white bars) at 4 and 8 months of age and (B) the density of ChAT-immunoreactive neurons per 100,000 µm2 within these two areas, expressed as a percentage of controls. The MSN and VDB area measurements were performed in the dorsoventral and mediolateral aspects of each nucleus on five sections using the NIH Image system with a scale bar corrected to actual size and anatomical landmarks described under Materials and Methods section. As can be seen in (A), there was no difference in terms of total area of the nucleus between trisomics and controls at any given age or area. However, there was a significant decrease in the density of ChAT-immunoreactive neurons within both the MSN (P , 0.05) and VDB (P , 0.001) in the trisomic animals (black bars) and age-matched controls (white bars). (B) Reflects the pooled values of 6-, 8-, and 10-month-old animals for each group, giving a final n of 9 per group.

high-affinity NGF receptor, trkA. A significant decrease in the density of trkA-positive neurons was observed at 6, 8, and 10 months of age in the Ts65Dn mice, compared to age-matched controls (see Figs. 6–8). Microscopical observation demonstrated a significant shrinkage of trkA-immunoreactive neurons in both MSN and VDB. Close inspection of the neurons revealed a distribution difference in trkA-positive vesicles within the cytoplasm of the different groups, so that the

neurons in young animals (4 months old) and older control animals demonstrated a more even distribution of trkA immunoreactivity, compared to the Ts65Dn mice that were 6 months and older (see Fig. 6). The basal forebrain neurons in older Ts65Dn mice exhibited a patchy immunostaining for trkA, with clumps of intense labeling primarily in areas surrounding the nucleus, and less prominent labeling in the distal cytoplasm and in neurites (see Fig. 7). This interesting observation may relate to differences in cell function and/or transport of the trkA protein within the cholinergic neurons as they are gradually losing their phenotype. When image analysis of trkA-immunoreactive cell body size was performed, we found significant decreases in trkA cell size in trisomic animals at 6 months (P , 0.01, n 5 3 in each group), 8 months (P , 0.01; n 5 3 per group), and 10 months (P , 0.05; n 5 3 per group) months of age in the MSN of Ts65Dn mice, compared to controls (see Fig. 8). A significant cell shrinkage was also observed in trisomics in the 8-month group in the VDB (Fig. 8C; P , 0.05). The mean values for each of the eight groups are shown in Figs. 8A and 8C. The shrinkage of trkA-immunoreactive cells was less notable in the nucleus basalis, with an average shrinkage of 16% in the Ts65Dn mice at 10 months of age. Even though the density of trkA-immunoreactive neurons was significantly decreased, and the cells appeared shrunken and atrophied, it was surprising to note that there was not a significant decrease in the staining intensity for trkA within the remaining neurons in either of the areas examined (see Figs. 8 B and 8D). There was a significant variability in the staining intensity both within groups and within each animal, but the neurons appeared to stain as densely for trkA in both groups, even up to 10 months of age. DISCUSSION

The present paper demonstrates that mice with a segmental trisomy of the distal portion of chromosome 16 (Ts65Dn) undergo a partial degeneration of cholinergic basal forebrain neurons and a decrease in performance on spatial, but not visual reversal learning between 4 and 8 months of age. Cresyl violet-stained sections did not reveal any active cell death processes or overall cell loss in Ts65Dn mice at any of the ages studied, at least not upon gross morphological evaluation (Fig. 2). Had there been a massive cell death in the MSN or VDB area at the age of 6 months in the Ts65Dn mouse forebrain, this process would have been obvious with fragmented nuclei and numerous extracellular

FIG. 6. Immunohistochemical labeling of trkA receptors in basal forebrain of Ts65Dn mice (labeled with ‘‘Ts’’ on the left) and controls (labeled with ‘‘C’’ on the right). a and b, 4 months old; c and d, 6 months old; e and f, 8 months old; and g and h, 10 months old. The age in months for each row is shown in the bottom left corner. Note the gradual decrease with age in the Ts group (left) also with this marker in the VDB area shown here. Similar results were obtained in the MSN area. Bar in h, 75 µm.

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FIG. 7. High-magnification micrographs of medial septal nucleus neurons labeled with trkA antibodies. a and b, 4 months old; c and d, 6 months old; and e and f, 10 months old. The age in months is indicated in the bottom left corner of each row. The left side of the figure is sections from Ts65Dn mice (Ts), and the right side is figures from controls (C). Note the difference in cell size as well as staining distribution between the trisomics and controls at 6 and 10 months (c–f). Vesicles containing trkA labeling are seen in close proximity to the nucleus (arrows) in trisomic animals, while the neurons in control animals exhibit a more even distribution of immunoreactivity. In addition, labeling of neurites, at least proximal to cell bodies, is evident in the later stage controls (d and f), but not in 6- or 10-month-old Ts65Dn mice (c and e). Bar in f, 30 µm.

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FIG. 8. Bar graphs illustrating the average cell sizes in square micrometers (A and C), as well as staining intensity (expressed as a staining: background value on a relative scale from 0 to 256) within each neuron (B and D) with trkA antibodies, in MSN (A and B) versus the VDB (C and D). White bars, controls; black bars, Ts65Dn mice. Note the gradual decrease in cell size in the Ts65Dn mice at 6–10 months, both in the MSN and in the VDB. Statistical analysis with ANOVA and Tukey–Cramer post hoc test revealed significant differences between the two groups at 6 months (P , 0.01), 8 months (P , 0.01), and 10 months (P , 0.05) in MSN and at 8 months (P , 0.05) in the VDB. There were no differences between the two groups in terms of average staining intensity for trkA within the neurons, either in the MSN or in the VDB, at any of the time points examined (B and D).

debris within this specific region. The loss of trkA and ChAT immunoreactivity occurred between 4 and 6 months of age, since the neurons were within normal range in the set of animals studied at 4 months of age. This finding correlated well with the behavioral studies, since the Ts65Dn mouse does not exhibit significant memory loss until 5–6 months of age (12). The packing density of ChAT-immunoreactive neurons was severely affected already at 6 months of age, and this loss was maintained throughout the experiment (10 months). In addition, the neurons exhibited significant shrinkage in mean cell body size and a moderate decrease in ChAT immunostaining intensity, even though the staining intensity was very variable both within animals and groups. There was no alteration in total volume of the basal forebrain in Ts65Dn mice (see Fig. 5A), thus indicating that the effects seen on neurons per section were not derived from simply an increase in total volume of the nucleus with age.

Our data are in line with previous findings by Megias et al. (43), since they found no cholinergic deficits in the basal forebrain at the age of 3 months in the Ts65Dn mice. Thus, the age-related deficits in this transmitter system reported by others previously (35) do not occur during development, but rather later in life, similar to what can be seen in the DS and the AD patients. Holtzman and collaborators (35) have performed a detailed stereological cell count of p75 (low-affinity NGF receptor)-immunoreactive neurons in the forebrain of Ts65Dn mice at 10 weeks, 6 months, and 20 months of age. They found a cell loss of 30% in the forebrain at 6 months and 40% at 20 months of age, compared to age-matched controls. These values are comparable to the values described in the present study, since we found a cell loss of 31% in the MSN and 48% in the VDB, respectively. It is comforting to note that the values were so similar, particularly since the two studies were performed using two completely differ-

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ent image analysis methods and in two independent laboratories. The mean cell size measurements were almost identical in their study (35) compared to ours as well, which further supported our findings. Even though we did not perform stereological analysis in our laboratory, area measurements demonstrated identical volumes for both MSN and VDB between the two groups, at least at 4 and 8 months of age (Fig. 5A). Thus, the cell packing density values presented in Figure 5B could not solely depend on a volume difference between Ts65Dn mice and wild-type mice, causing the neurons to be more dispersed in one group or the other. There is an ongoing debate regarding the unbiased stereology method, and our results presented here directly demonstrate that stereological measurements can give similar outcomes as a more traditional analysis method in terms of cell loss in a particular area, at least in this mouse model. The fact that reversal learning is more impaired than initial discrimination learning in the Ts65Dn mice indicates that forebrain structures underlying behavioral flexibility and inhibition of behavior are likely impaired. Further, because this deficit exists only for the spatial and not the visual discrimination reversal tasks it indicates likely involvement of the hippocampus in this deficit. The significant effect of age upon spatial reversal suggests that the loss of medial septal cholinergic neurons may contribute to the behavioral deficits that develops after 6 months of age in the Ts65Dn mice. This same deficit has been demonstrated using shock escape as reinforcement (12). The increase in arm entries seen in trisomic mice typically consisted of repeated entries into the incorrect arm, followed by returns to the start arm and reentries into the incorrect arm and thus truly demonstrates perseveration in responding to the previously rewarded side of the maze and thus a deficit behavioral flexibility that was specific to the spatial task. A much more severe cholinergic deficit was observed already during development in mice that had a trisomy of their entire chromosome 16 (Ts16; see 24). It was suggested by Nelson and collaborators (50) that the cholinergic deficits seen in Ts16 mice could be due to either a lack of supportive factors or additional inhibitory factors produced by glial cells in the same mouse, since cultured cholinergic neurons from Ts16 mice survived and expressed cholinergic phenotype when grown together with wild-type glial cells instead of trisomic glia. Further evidence for the trophic theory of degeneration in these neurons was provided by Holtzman and collaborators (33) who treated cholinergic neurons from Ts16 mice with NGF and found a significant enhancement of survival and expression of cholinergic markers. To our knowledge, NGF rescue therapy has not been attempted for the cholinergic deficits seen in Ts65Dn mice, even though our findings with the

decrease in staining for the high-affinity NGF receptor trkA (Figs. 6 and 7) suggest that decreased NGF function may be a biological mechanism for the cholinergic neuron atrophy also in the Ts65Dn mouse. The difference between this mouse model and previous animal models, using, for example, age-related or excitotoxically induced cholinergic cell death, is that the Ts65Dn mouse exhibits increased gene dosage of regions of chromosome 21 homologous genes that are present in DS, allowing analysis of the role of these genes in cholinergic neuron loss. The particular deficiency in NGF release, uptake, or transport seen in patients with AD, or in animal models of this condition, has not yet been revealed. One crucial question that arises from these studies is whether the loss of nerve growth factor precedes the loss of cholinergic phenotype in the forebrain, as suggested by most investigators, or if the NGF receptor loss described is a result, and not a cause, of this condition (see, e.g., 63). In aged rodents, a deficiency in the retrograde transport of NGF has been suggested to be directly related to programmed cell death in the basal forebrain. Cooper et al. (7) demonstrated a significant reduction in the number of cholinergic neurons that had maintained an intact retrograde NGF transport system in the aged rat, and they also demonstrated that those neurons that did not transport NGF well also were the ones that underwent shrinkage and loss of phenotype in the forebrain. Taken together, findings reported in AD patients and animal models of this disease indicate that loss of trkA receptor binding to NGF and subsequent decreases in retrograde transport to the cell body may be causative factors in degeneration of cholinergic neurons, and the findings reported here support this theory also for Ts65Dn mice. There was a significant difference in distribution of trkA immunoreactivity in the forebrain neurons of Ts65Dn mice compared to age-matched controls, even though image analysis did not show differences in mean staining density between the groups. The trkA immunoreactivity was distributed in clumps surrounding the nucleus in trisomic mice, and very few neurites were labeled at the later time points (6 months and older). The unaltered trkA staining intensity within each surviving neuron in Ts65Dn forebrain suggests that the neurons that had survived the events occurring during months 6–10 in the Ts65Dn mice could have survived because of their unaltered trkA content. Other studies have shown that cell body size is a sensitive measure for the functional state of forebrain cholinergic neurons (31), thus substantiating the findings in our study that segmental trisomy of mouse chromosome 16 leads to significant decrease in average cell body size of these neurons, suggesting indeed that these neurons are dysfunctional in the Ts65Dn mouse after 6 months of age. It has been suggested that exogenous NGF administration may

CHOLINERGIC PHENOTYPE LOSS IN A MURINE DOWN’S SYNDROME MODEL

enhance activity and/or synthesis of the high-affinity trkA receptor and thus increasing the utilization of endogenous NGF release and/or uptake in the target region. Conversely, multiple studies using both lesions and aged animals have demonstrated that cholinergic degeneration is coupled with a decrease in NGF receptors, again suggesting a close link between the well being of these neurons and utilization of NGF. The hippocampal target area has also been found to be defective in Ts65Dn mice. For example, it was shown that hippocampal slices from both young and aged Ts65Dn mice exhibited a significant decrease in longterm potentiation (LTP; see 60). These authors suggested that the decrease in LTP might be, at least in part, responsible for the behavioral deficits in terms of memory and learning observed in these mice. Structural alterations in the hippocampal formation occur in the Ts65Dn mice, and it is not clear whether the cholinergic deficit is the cause or effect of these hippocampal alterations. For example, increased levels of the amyloid precursor protein (APP) have been demonstrated in the hippocampal formation of the Ts65Dn mice (54), and a significant loss of neuronal density was observed in the CA3 region of the hippocampus of these mice as well (36). Thus, careful examination of the time course of these different alterations is needed in order to determine whether hippocampal damage is caused by cholinergic degeneration or the other way around. Since a similar parallel time course of hippocampal/ cortical/forebrain damage appears to happen in the AD patient during progression of the disease (see, e.g., 17, 51, 66, 68), the Ts65Dn mouse may be used as a model system to determine the role of the target and the innervating neuronal phenotype in the septohippocampal pathway. It has not yet been demonstrated which specific genes on the triplicated segment of Chr 16 of the Ts65Dn mouse that may underlie the time-dependent cholinergic phenotype loss described here. However, further evaluation of potential candidate genes in this particular portion of the murine Chr 16 has been performed by Sago et al. (55), who have recently reported that Ts108Cje, a shorter trisomy on mouse chromosome 16, results in learning deficits to a lesser degree than the Ts65Dn mice. In addition, they reported that the cholinergic deficits seen in Ts65Dn mice and reported here were absent in the Ts108Cje mice. This presents an important opportunity to explore alterations in specific gene expression in the Ts65Dn mice and could potentially lead to candidate genes also for AD and DS. It is interesting to note that one of the genes that is missing on the segment triplicated in the Ts108Cje mouse, but present in the Ts65Dn mice studied here, is the gene for the APP (see 55). APP is an integral protein with soluble derivatives that has been suggested to be intricately linked to the pathology of

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AD (29). Interestingly, mutations of APP itself, or the presinilins leading in turn to APP alterations, have been distinctly linked to early-onset familial AD (e.g., 5, 18, 66), which is the form of AD that occurs in DS patients. This gene is therefore strongly implicated in the process of cholinergic degeneration also in our mouse model. APP has been linked to the trophic actions of NGF. For example, it has been demonstrated that APP administration to PC12 cells results in an increase in neurite outgrowth and branching, and application of APP antibodies to cultured PC12 cells results in a blockade of NGF effects (44). Other studies have shown that b-amyloid (a product of APP cleavage) can interact with the binding properties of low-affinity NGF receptors (69). Even though we may be able to detect candidate genes on mouse chromosome 16 that appear to be important for cholinergic degeneration, caution must be taken when translating these results to patients with DS and AD. For example, overexpression of genes on the 21st chromosome in DS patients may alter expression of genes on other chromosomes (see, e.g., 1, 11), and genes on the 21st chromosome may interact with each other to give rise to the degenerative changes observed. In conclusion, the studies presented here demonstrate that the Ts65Dn mouse is a model in which cholinergic forebrain neurons undergo alterations during adult life. The parallel loss of cognitive function in the mice and subsequent decrease in high-affinity NGF receptors suggest that the model may be used to develop future treatment therapies for patients undergoing a similar spontaneous loss of cholinergic phenotype in the forebrain. ACKNOWLEDGMENTS The immunohistochemical studies were supported by USPHS Grants AG12122, AG04418, AG15239, and AG10755, and the behavioral studies were supported by a Mental Retardation Research Center Grant (HD04024) and a Down syndrome Program Project Grant (HD17449). A.-C.G. was supported by a career development award (Grant AG00796). We thank Dr Louis Reichardt for the generous gift of trkA antibodies. In addition, we thank Muriel T. Davisson for the gift of breeding stocks; David Patterson, Iris Hart, and Lynne Melteson for typing the mice; and Berit Engst, Joo Kwon, and Joan Ivaska for technical assistance.

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