Effects of long-term memantine on memory and neuropathology in Ts65Dn mice, a model for Down syndrome

Abstract

Memantine is a partial NMDA receptor antagonist that has been shown to improve learning and memory in several animal models, and is approved for the treatment of Alzheimer's disease (AD). Chronic treatments using memantine in animal models of Alzheimer's disease show disease-modifying effects and suggest a potential neuroprotective function. The present study assessed the effects of both short- and long-term memantine treatment in a mouse model of Down syndrome (DS), the Ts65Dn mouse. The Ts65Dn mouse contains a partial trisomy of murine chromosome 16, and exhibits hippocampal-dependent memory deficits, as well as progressive degeneration of basal forebrain cholinergic neurons (BCFNs). Ts65Dn mice were treated with memantine for a period of 6 months, beginning at 4 months of age. At the end of treatment the mice underwent memory testing using novel object recognition and water radial arm maze tasks, and then histologically analyzed for markers of neurodegeneration. Memantine treatment improved spatial and recognition memory performance in the Ts65Dn mice, though not to the level of normosomic littermate controls. Despite these memory improvements, histological analysis found no morphological signs of neuroprotection of basal forebrain cholinergic or locus coeruleus neurons in memantine-treated Ts65Dn mice. However, memantine treatment of Ts65Dn mice gave rise to elevated brain-derived neurotrophic factor expression in the hippocampus and frontal cortex, suggesting a mechanism of behavioral modification. Thus, our findings provide further evidence for memory facilitation of memantine, but suggest pharmacological rather than neuroprotective effects of memantine both after acute and chronic treatment in this mouse model.

Copyright © 2010 Elsevier B.V. All rights reserved.

Figures

Figure 1

Study timelines. Study 1 consisted of a long-term oral memantine treatment in which Ts65Dn mice and normosomic controls (NS) received memantine (20 mg/kg) for 6 months, beginning at 4 months of age. Mice from Study 1 were first tested on a 3-day win-stay RA maze at 8.5 months of age. Following spatial memory testing, the mice underwent a series of four weekly NOR testing sessions. Prior to the 3rd NOR testing session, mice that had previously received memantine were switched to control fluids, such that the 3rd session occurred with mice “off” treatment. Following this session, mice were returned to their treatments for session 4. Study 2 evaluated the effects of acute memantine administration on recognition memory in Ts65Dn mice, as naïve Ts65Dn mice received acute memantine injections (10 mg/kg, i.p.) immediately prior to NOR training and testing periods.

Figure 2

Spatial memory deficits were attenuated following memantine treatment in Ts65Dn mice. A, Activity was measured over a one-hour session (distance traveled, cm). Ts65Dn Control and Ts65Dn Mem mice show greater spontaneous activity than NS Control and Mem mice. Memantine treatment did not significantly affect either genotype (Genotype effect: **p<0.01). B, Performance in a win-stay RA maze task is indicated by errors per trial across days. NS Control mice show reduced errors across days. While Ts65Dn Control mice performed significantly more errors on days 2 and 3 than NS Controls, Ts65Dn Mem mice show a reduction in errors relative to Ts65Dn Controls. Memantine treatment had no effect on NS mice (Days 2–3: *p<0.05 compared with NS mice, ^p<0.05 between Ts65Dn Mem and Ts65Dn control groups; mean ± SEM).

Figure 3

Memantine-treatment recovered novel object recognition in Ts65Dn mice following both chronic and acute delivery. A, Novel object recognition performance, as shown by discrimination indices (DI), indicate Ts65Dn did not discriminate object novelty, showing no preference for novel objects and a reduced DI compared to NS control mice. Ts65Dn Mem mice, in contrast, were able to discriminate object novelty. B, Total exploration time was not altered by memantine-treatment (n = 10–14). C, A cross-over trial was carried out in the memantine-treated mice to determine whether memantine-treatment would sustain performance following removal of the drug. Following memantine removal of one week, these Ts65Dn mice were no longer able to discriminate object novelty, while reinstatement of memantine recovered their preference for novel objects (*p<0.05 compared with NS mice; mean ± SEM).

Figure 4

Memantine-treatment did not prevent cholinergic neuron atrophy in basal forebrain. A–C show TrkA-positive cholinergic neurons in the medial septal nucleus in NS control (A), Ts65Dn control (B), and Ts65Dn Mem (C). Arrows show normal BFCN phenotype, while arrowheads indicate smaller neurons exhibiting less robust TrkA-immunoreactivity (40x magnification, scale bar = 40 µm). D, Cell size measurements depict significant reductions in cell area in control and memantine-treated Ts65Dn mice relative to NS (*p<0.05).

Figure 5

Locus Coeruleus degeneration in Ts65Dn mice was not altered following memantine administration. A–C, TH immunostaining of the rostral pons illustrates reduced fiber density and neuron number in Ts65Dn control (B) and Ts65Dn memantine (C; scale bar = 100 µm) compared to NS mice (A). D, Stereologic measurements of TH-positive cells in the Locus Coeruleus confirm a reduction in LC neurons in both Ts65Dn control and memantine mice (**p<0.01).

Figure 6

Cal 28k, a calcium binding protein lost early in Ts65Dn mice, was not altered following memantine-treatment. A–C, Immunohistochemical analysis of hippocampal neurons in the CA1 reveals Cal 28k immunoreacivity in the cell bodies and apical dendrites of pyramidal cells in NS mice (A). Hippocampal neurons of Ts65Dn control (B) and memantine (C) mice show fewer immunoreactive pyramidal neurons and dendrites (scale bar = 100 µm). D, Densitometry revealed that NS mice demonstrate significantly higher Cal 28k-immunoreactivity than Ts65Dn groups (**p<0.01; Immunoreactivity normalized to background; mean ± SEM).

Figure 7

Microglial activity was not reduced in the hippocampus of Ts65Dn mice following memantine treatment. A–C, CD45-positive microglia in the CA1 of the hippocampus exhibit lower immunoreactivity indicative of a “resting” state in NS Control mice (A). Ts65Dn control (B) and memantine-treated (C) mice show larger microglia with greater staining density, consistent with an increased microglial activity (scale bars = 100 µm, 20 µm for inset). D, Microglial immunoreactivity in the hippocampus is elevated in Ts65Dn mice, and is not affected by memantine-treatment (*p<0.05; Immunoreactivity normalized to background; mean ± SEM).

Figure 8

Memantine increased BDNF gene expression in the hippocampus of Ts65Dn mice. A, Ts65Dn Mem mice exhibited higher BDNF mRNA levels in the hippocampus compared to normosomic controls (p<0.05), while NS Mem mice did not differ from NS control mice. B, Protein measurements in frontal cortex tissue showed a significant increase in BDNF levels in Ts65Dn Mem mice relative to NS controls. This increase in BDNF was not observed in NS Mem mice (*p<0.05; Mean ± SEM).

Figure 9

Acute memantine restored object discrimination in Ts65Dn mice. In study 2, a separate group of mice underwent acute memantine injections immediately prior to training and testing phases. Acute memantine treatment effectively recovered novel object preference in Ts65Dn mice. *p