Research report
Evaluation of the APP23-model for Alzheimer's disease in the odour paired-associate test for hippocampus-dependent memory

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Abstract

The APP23 model is a transgenic mouse model for Alzheimer's disease. Cognitive performance in the APP23-model was assessed by Morris Water Maze (MWM) and passive avoidance learning, but the latter failed to show any difference between the genotypes. In search of a non-spatial alternative for assessment of hippocampus-dependent memory, we evaluated an odour paired-associate test, which is based on learning an association between two sets of odours. The protocol includes a shaping phase, in which the animals learn to dig up a reward, a preliminary training phase and a training phase, where the actual association is learned. Subsequently, mice are tested for transitive inference and subjected to a symmetry test. Impairment was seen in the APP23 mice, in comparison with wild type mice, in training; however, both groups failed the transitivity and symmetry test. Possible explanations for this discrepancy with earlier published results are the advanced age of the mice or the C57Bl/6J background, in which the model was established.

Introduction

Alzheimer's disease (AD), the most common form of dementia, is characterised by progressive cognitive impairment and behavioural disturbances. Mutations in the amyloid precursor protein (APP) are associated with autosomal dominant AD. Transgenic mouse models expressing wild type or mutated human APP reproduce some of the neuropathological features of AD, as well as behavioural symptoms (for review see [28]). In this experiment we used the APP23 model [24]. This transgenic model expressed 751-amino acid human APP (hAPP) with the Swedish double mutation (K670N/M671L), driven by the neuron-specific murine Thy1-promotor, which results in a sevenfold overexpression of hAPP compared to endogenous APP. The brains of these mice contained neuritic plaques, microglial reactivity and hyperphosphorylated tau from the age of 6 months onwards [3]. A 10% cell loss in pyramidal neurons of the CA1 hippocampal region was found at the age of 14–18 months, whereas quantitative cell loss in the entorhinal cortex could not be assessed because the massive amyloid deposition in aged APP23 mice prevented the reliable identification of the anatomical borders [2]. Nevertheless, qualitative neuronal loss was clearly observed in the entorhinal cortex of aged APP23 mice [6]. Basic synaptic function in the hippocampus was reduced in APP23 mice compared with their wild type littermates at 12 and 18 months of age, whereas long-term potentiation, a cellular model for memory, was unaltered [21]. In our experiment we included old animals to be certain of hippocampal cell loss and synaptic alterations at the moment of testing for transitivity and symmetry. In previous experiments impairment of memory in the APP23 model in various circumstances was already established. Significant visual spatial learning difficulties were observed from the age of 3 months onwards in the APP23 model using the Morris Water Maze [8], [14], [16], [27]. Passive avoidance learning in a step-trough box showed that male and female APP23 mouse were not impaired in this test until the advanced age of 25 months [14], [27]. In a plus-shaped water maze, impairment of spatial working memory was demonstrated [29]. Wild type and APP23 animals performed equally well in radial maze, but wild type mice performed better in the complex maze [13]. A modest deficit in spatial learning was shown during the initial learning phase of the Barnes maze test in APP23 mice and there were indications for a deficit in the use of spatial search strategies in APP23 mice [20].

Until now, cognitive assessment in the APP23 model has only focussed on spatial memory abilities and passive avoidance learning. Given the fact that the hippocampus may also be involved in non-spatial memory [1], [10], [15], we decided to subject the APP23 model to a non-spatial hippocampus-dependent learning paradigm. We chose an odour-guided paired-association task [5], analogous to the verbal paired-association task for humans [31].

An advantage of this test is the fact that the mice do not need to swim. Provided the animals can dig, motor impairment will not influence the results. Moreover, the results are not biased by anxiety, since the test is based on natural scavenging habits, in contrast to the Morris Water Maze paradigm. In addition, the paired-associate task can form a solution for the assessment of memory in transgenic mice with impaired vision based for example on the presence of a recessive mutation in the background strain causing retinal degeneration [19].

In order to exclude the possibility of a progressive development of hyposmia, we investigated the olfactory sense in the APP23 model at the age of 75 weeks. Based on a novel odour test, olfactory acuity was already proven to be intact in male APP23 mice at the age of 6–8 weeks, 3, 6 and 12 months [30].

Section snippets

Transgenic mouse model

APP23 mice were generated and bred as previously described [24], and backcrossed to the C57BL/6J strain for more than 20 generations to create an isogenic line. Experimenters were blinded as to the genetic status of the animals. Genotypes were elucidated by polymerase chain reaction. All experiments were carried out in compliance to the European Communities Council Directive (86/609/EEC) and all protocols were approved by the Local Animal Ethics Committee.

Novel odour test

Olfaction was investigated using the

Novel odour test

No differences between genotypes in the latencies to the first entries in the target circle (near the odorant) or in entire the target zone (t-test; P = 0.348 and P = 0.825) were observed. Genotype groups did not differ in the total time spent in the target circle (P = 0.912) and the target zone (P = 0.540). We did not find an effect of genotype on the ratio of number of entries in the target circle (P = 0.971) or in the target zone (P = 0.603) over the total number of entries.

Food restriction

During the whole experiment,

Discussion

Based on previous results [30] supplemented with our data from the novel odour test, we can conclude olfactory sense is intact in APP23 and wild type mice until the advanced age of 75 weeks, by which a necessary prerequisite for an olfactory based memory test is fulfilled.

In the preliminary training, neither genotype showed a preference for a specific odour during preliminary training, thereby excluding preference bias in subsequent testing phases.

The aim of the paired-associate task was the

Acknowledgements

This work was supported by the Fund for Scientific Research-Flanders (FWO grant G.0038.05), Stichting voor Alzheimer Onderzoek (SAO) – Fondation pour la Recherche sur la Maladie Alzheimer (FRMA), Interuniversity Poles of Attraction (IUAP Network P6/43), agreement between Institute Born-Bunge and University of Antwerp, the Medical Research Foundation Antwerp, the Thomas Riellaerts research fund, and Neurosearch Antwerp. D.V.D. is a postdoctoral fellow of the FWO.

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