In this study, we evaluated the effects of chronic memantine administration on amyloid plaque deposition and neuronal morphology using quantitative light and electron microscopy in Tg2576 mice, a commonly used animal model of AD. In addition, we sought to evaluate the behavioral consequences of any observed changes in structure by assessing contextual memory using a fear-conditioning paradigm. We found that chronic memantine administration decreased Aβ plaque burden in the brain, increased synapse density and increased degenerating axon density in the molecular layer of the dentate gyrus. While we didn't observe neuronal vacuolization following memantine administration, the appearance of degenerating axons after memantine administration was dose-dependent and consistent with some of the neurotoxic changes previously observed following administration of NMDA antagonists. However, despite these changes in amyloid deposition and neuronal structure, we did not find an effect of memantine on hippocampal-dependent contextual memory in Tg + mice.
Our results are consistent with previous studies which found decreases in cortical APP levels in Tg2576 mice after 10 days of memantine treatment (Unger et al, 2006
). However, the mechanism by which memantine regulates amyloid metabolism is not clear. It has been shown that increased neuronal activity leads to increased APP production, a likely precursor to Aβ
plaque formation (Cirrito et al, 2005
). Thus, blockade of NMDA receptors by memantine could reduce neuronal activity and subsequently lower APP production. In support of this hypothesis, we noticed that the lower dose of memantine (5 mg/kg) exerted stronger effects in reducing Aβ
plaque number and burden than the two higher doses (10 mg, and 20 mg/kg) (). Thus, it is possible that lower memantine doses may block just enough neuronal activity to lower amyloid production, while higher doses block so much neuronal activity that APP degradation and clearance mechanisms are also decreased. Our experimental design was such that the animals began treatment before the appearance of plaques would be expected to occur in the brain. Thus, our results cannot address the question whether memantine has the capacity to prevent the formation of new plaques or to reduce the number of Aβ
plaques already formed.
In our study, we found that memantine administration increased synaptic density in the molecular layer of the dentate gyrus in both Tg + and Tg− mice; in fact, memantine-treated Tg + mice tended to have a greater quantity of synapses than vehicle-treated Tg− mice. The molecular layer of the dentate gyrus is one of the first regions to show amyloid deposition and synapse loss in the Tg2576 mouse model of AD (Su and Ni, 1998
; Reilly et al, 2003
; Dong et al, 2007
). However, it is difficult to know the implications of increases in synapse density after memantine administration in Tg2576 mice. While this could represent a neuroprotective effect, it could also be compensatory to memantine-induced axonal degeneration. Since too many as well as too few synapses could adversely impact neuronal function, caution should be used in interpreting the observed changes in synapse density after memantine treatment.
We observed no neuronal vacuolization or other histopathological changes in the hippocampus, posterior cingulate and retrosplenial cortices at the light microscopy level after memantine administration as previously reported after the administration of NMDA antagonists (Fix et al, 1993
; Creeley et al, 2008
). However, our observation that chronic memantine administration was associated with axonal degeneration in the hippocampus may relate to other reported neurotoxic effects of NMDA antagonists (eg PCP, MK-801) (Low and Roland, 2004
). The use of NMDA antagonists as therapeutic agents for neuropsychiatric disorders thought to include a component of neurodegeneration has been investigated for many years. However, their use for this purpose has been hampered by observations of adverse behavioral effects such as hallucinations and memory disturbances in humans, and reports of neuronal vacuolization, neuronal and axonal degeneration, and induction of heat-shock protein in rodents (Olney et al, 1989
; Sharp et al, 1992
). Memantine, at doses somewhat higher than the doses used in this study (50 mg/kg), has been shown to produce neurodegenerative effects in the rat (Creeley et al, 2006
). While the axonal degeneration induced by high doses of memantine may occur because of direct effects on neurons, indirect effects on oligodendrocytes may also be possible. Notably, NMDA receptors are ubiquitous within the CNS and are expressed not only on neurons, but also on glial cells (for review see Verkhratsky and Kirchhoff, 2007
). Oligodendrocytes are important for the survival as well as function of neurons (Lappe-Siefke et al, 2003
; Popko, 2003
), and thus, blockade of NMDA receptors on oligodendrocytes could eventually result in axonal degeneration. Our data lend preliminary support for this hypothesis in that we observed occasional degenerated oligodendrocyte somas in memantine-treated animals ().
We were surprised to find no effect of memantine on contextual memory in a fear conditioning paradigm given that we used doses similar to those previously been reported to have beneficial behavioral effects in other transgenic mouse models of AD (Minkeviciene et al, 2004
; Van Dam et al, 2005
; Van Dam and De Deyn, 2006
). The behavior deficits we observed in Tg + mice replicated earlier findings using Tg2576 mice (Hsiao et al, 1996
; Corcoran et al, 2002
; Arendash et al, 2004
; Barnes and Good, 2005
; Dong et al, 2005
) and thus imply validity of the behavioral test employed here. At present, the effects of memantine on behavior in transgenic mouse models of AD (Minkeviciene et al, 2004
; Van Dam et al, 2005
; Van Dam and De Deyn, 2006
), as well as other models (Barnes et al, 1996
; Miguel-Hidalgo et al, 2002
; Lang et al, 2004
; Woodruff-Pak et al, 2007
; for review see Yuede et al, 2007
) are inconsistent. Minkeviciene et al (2004)
reported improved acquisition in water maze using APP/PS1 transgenic mice treated with 30 mg/kg memantine in drinking water for 3 weeks, whereas no effect was observed on retention. Conversely, Van Dam et al (2005)
reported beneficial effects of a much lower dose (2.0 mg/kg) of memantine while higher doses (10 mg/kg) demonstrated detrimental effects in the water maze. As compared with these studies, we found that a 5 mg/kg dose was associated with better performance than a 10 mg/kg dose, even though we did not detect a significant overall effect of memantine in Tg2576 mice. Explanations for these inconsistencies include the selection of the particular mouse model, the drug doses, or the duration and route of drug dosing.
The inconsistency between our morphological and behavioral results highlights the difficulty of defining the structural basis of behavioral deficits in AD mouse models. Previous studies showed little or no correlation between the quantity of Aβ
deposition and behavioral deficits (Terry et al, 1991
; Arriagada et al, 1992
; Berg et al, 1993
; Westerman et al, 2002
; Van Dam et al, 2003
) in both humans with AD and animal models of AD. Increasing evidence has indicated that soluble amyloid oligomers may be directly toxic to neuronal structure and function (Gong et al, 2003
; Watson et al, 2005
; Lacor et al, 2007
; Shankar et al, 2007
). Another explanation of the inconsistency between structure and behavior observed in this study is that the animals may have developed adaptive or compensatory responses to memantine over the extended administration period. In such a scenario, the benefits of memantine on plaque burden and synapse density might have been insufficient to produce persistent behavioral changes. Finally, it is also possible that the conditioned fear paradigm used in this study was not sensitive enough, or our sample sizes were too low to detect small behavioral changes with memantine administration.
If memantine has the potential to produce both beneficial and detrimental effects in the mammalian brain, the task is to find a dose of memantine with the former but not the latter properties. We selected three doses (5, 10, and 20 mg/kg) for this study in an attempt to approach the therapeutic doses typically used in humans (Danysz and Parsons, 2003
, Wenk et al, 2006
). Previous studies in mice have reported that a 30 mg/kg oral dose of memantine in drinking water produces a steady-state plasma drug level of around 1 μM, which is thought to be therapeutic based on clinical studies (Kornhuber and Quack, 1995
; Minkeviciene et al, 2004
). However, it is difficult to select drug doses in animal studies that are equivalent to clinical doses because the composition and responses observed in the brains of rodents and humans differ (Wenk et al, 2006
). Thus, our results should not be used to predict a dose of memantine in human subjects that is both safe and effective. Rather, our results suggest that in vivo
neuroimaging should be combined with clinical and cognitive assessments in an effort to determine the effects of such drugs on brain structure and function as well as symptomatology. More effective treatments are greatly needed for neurodegenerative disorders, such as AD, and the NMDA receptor remains a viable pharmacological target in these efforts. However, drugs that act on the NMDA receptor have the potential to damage neurons as well as to protect them, and investigations of such drugs should be performed with appropriate attention to this possibility.