In the current study, we assessed age-dependent electrophysiological and morphological properties of principle neurons within the lateral amygdala in male human apoE TR, apoE KO, and C57 mice. Our results revealed that the human apoE TR mice display differential effects on synaptic integrity compared to each other, as well as to C57 and apoE KO mice. First, expression of apoE4 results in significantly lower dendritic arbor and excitatory transmission compared to non-apoE4 mice by 7 months of age. Second, co-expression of apoE2 with apoE4 attenuates the loss in synaptic activity, but does not alter dendritic length. This result supports the hypothesis that expression of apoE2 has a protective effect on synaptic integrity. Third, the apoE KO mice display reduced excitatory transmission similar to apoE4 mice, while C57 mice are more similar to apoE2/4 mice at 7 months. Fourth, a significant reduction in excitatory activity is present in apoE4 TR mice as early as 1 month of age while the loss of dendritic arbor has not yet begun to manifest. Finally, all cohorts (except apoE3 mice) show significant reductions in excitatory transmission with age (i.e. 1 to 7 months).
Whole cell recordings in brain slices from 1 month old mice revealed significant differences in excitatory transmission (i.e. sEPSC interval) between apoE4 mice and all other cohorts examined (except apoE KO mice). Although the frequency of synaptic transmission was lowest in the apoE4 mice at 1 month, we did not detect any statistically significant difference in neuronal morphology compared to apoE2/4 or apoE3 mice. Dumanis et. al found significant differences in neuronal morphology (i.e. spine number) within cortical layers II/III of 1 month apoE TR mice (Dumanis et al., 2009
). This same study, however did not detect any morphological differences in the dentate gyrus at 1 month. Thus, the effect of APOE
genotype on neuronal morphology appears to vary from one brain region to another. Comparisons between 1 and 7 month old apoE2/4 and apoE4 mice showed an increase in the sEPSC interval and a decrease in dendritic arbor with no change in the apoE3 mice. We did not extend morphological analysis to the C57 or KO mice as no significant differences were observed in the sEPSC interval at 1 month when compared to apoE3 mice.
The apoE4-associated deficit in synaptic activity in the lateral amygdala is consistent with previous electrophysiological studies performed in the hippocampus of apoE TR mice. For example, LTP in the dentate gyrus was significantly lower in apoE4 mice compared to apoE3 and C57 mice at 2–4 months (Trommer et al., 2004
). In contrast, apoE4 mice displayed enhanced LTP in the CA1 field (Korwek et al., 2009
). These regional differences are likely due to the complex circuitry of the hippocampus, where the dentate and CA1 comprise distinct circuitries and differing synaptic connections with various brain regions.
The reduction in synaptic activity in apoE KO is similar to two previous reports showing synaptic alterations in the hippocampus (Krugers et al., 1997
) (Veinbergs et al., 1999
) however differs from two other studies which found no differences in LTP, behavior or neuronal morphology, between apoE KO mice and controls (C57 mice) (Anderson et al., 1998
) (Fagan et al., 1998
). We did not detect any difference in neuronal morphology in 7 month old apoE KO mice compared to C57 or apoE3 TR mice. It is possible that synaptic deficits that occur in apoE KO mice do not appear until later in life, as Ji et al found that spine density and dendritic length was reduced in apoE KO mice at 12 months (Ji et al., 2003
), although this finding is refuted by a study which found no differences between apoE KO and C57 mice at 12 months (Anderson et al., 1998
). These contrasting studies highlight the inherent problems associated with using apoE KO mice to study the function of human apoE isoforms, as compensatory changes in other proteins and the extreme hyperlipidemic phenotype of apoE KO mice make it difficult to draw comparisons with human apoE expressing mice. Analysis of C57 mice showed that they were most similar to apoE2/4 and E3 mice at 1 month; however, by 7 months the C57 mice showed a significant reduction in excitatory activity while the apoE3 mice remained unchanged. This indicates that murine apoE functions differently than human apoE3 in the mouse brain with respect to excitatory transmission in the amygdala. This is not surprising given that murine and human apoE are very distant in evolutionary terms (e.g. only 70% amino acid homology between mouse and human apoE) (Weisgraber, 1994
The two major findings from this study are; one, the age dependent increase in mean sEPSC interval in all cohorts except the apoE3 TR mice, and two, the attenuation of synaptic activity (mean sEPSC interval) by apoE2 in the apoE2/4 mice. These results indicate a significant APOE
genotype effect on synaptic transmission in the TR mice. Analogies can be drawn with human cognitive studies which show that both age and APOE
genotype have a significant impact on cognitive ability (Riley et al., 2002
) (Snowdon et al., 2000
). Our results also support the idea that the APOE
alleles function in a co-dominant manner, otherwise the phenotype in the apoE2/4 mice would be indistinguishable from the apoE4 mice. Our results also support human gene association studies for relative risk of AD between varying APOE
genotypes (Roses et al., 1995
). For example, APOE2/4
heterozygotes have a significantly lower risk of developing AD compared to APOE4/4
homozygotes (Saunders et al., 1993
) (Corder et al., 1993
In our earlier study (Wang et al., 2005
) we were able to show a correlation between reduced synaptic transmission and reduced dendritic arbor in the apoE4 mice at 7 months. Herein we found 1 month old apoE4 mice displayed reduced synaptic transmission compared to apoE2/4 and apoE3 mice, however we did not detect any differences in neuronal morphology between the three cohorts. Since we were unable to measure spines in the 1 month cohorts it is possible that varying numbers of spines could account for the difference in synaptic activity. We are encouraged by the study in similar cohorts of mice (1 month TR mice) which found varying spine counts, albeit in a different brain region (Dumanis et al., 2009
). We plan to use Golgi staining techniques in future studies to aid in determining the number of spines, since we were unable to reliably count spines in any of these cohorts due to technical limitations with imaging the biocytin-filled cells.
We were able to measure other characteristics of neuronal morphology, however, and confirmed our original finding in the 7 month cohort which showed significant differences between apoE3 and E4 mice. Analysis of the apoE2/4 mice, however, showed no difference in dendritic length compared to apoE4 mice, which again could be explained by varying spine number. In the current study we elected not to use apoE2 homozygous mice in order to avoid any potential confounding effects of hyperlipidemia on synaptic integrity (Sullivan et al., 1998
). Future analysis of functional and morphological outcomes using hemizygous apoE TR mice (i.e. E2/E0 and E4/E0) could provide insight into the individual contribution of each isoform; however the apoE2/0 mice would be expected to develop type III hyperlipidemia, a similar confounding variable present in apoE2 and apoE KO homozygous mice.
The mechanism responsible for the reduction in synaptic transmission associated with apoE4 is currently unknown, but several possibilities may account for this effect. The lateral amygdala receives excitatory/glutamatergic afferents originating from the cortex and thalamus (McDonald et al., 2002
) and age-dependent pruning of dendrites and spines in cortical and thalamic afferents may contribute to the loss of excitatory activity. Since loss of synaptic integrity within the cortical limbic system is thought to begin very early in AD, potential disruption of these connections in the apoE4 TR mice could explain the reduction in synaptic transmission. While inhibitory inputs can also impact the degree of excitatory activity, our previous report showed no differences in the amplitude or frequency of spontaneous inhibitory synaptic currents (sIPSCs) between apoE3 and E4 (Wang et al., 2005
). It is plausible that the reduction in sEPSCs observed in apoE4 mice is due to the concomitant decrease in dendritic length of the post-synaptic cell. However, this does not explain our result seen in apoE2/E4 mice.
We had previously suggested that one explanation for the observed apoE4 deficit (at 7 months) was due to a deficiency in lipids required for neuronal remodeling (Wang et al., 2005
). The idea that apoE might regulate synaptic transmission via the distribution of lipids in neuronal membranes was first proposed by Mahley et al. (Mahley, 1988
) and Poirier et al. (Poirier, 1994
). It is now known that synaptic plasticity depends on glial derived cholesterol (Goritz et al., 2002
) which is delivered by apoE-containing HDL particles, and that phospholipids are required for maintaining synaptic plasticity (Koudinov and Koudinova, 2001
) (Hering et al., 2003
) (Bourre et al., 1989
) . Therefore, apoE4 (relative to apoE3 and E2) may be deficient in delivery of essential lipids for maintaining synaptic plasticity.
The observed apoE4 deficit in synaptic integrity could also be due to insufficient levels of functional apoE4 protein which would result in a rate-limiting supply of lipids required for synaptic plasticity. We and others recently showed that protein levels are lowest in the hippocampus and cortex of 3–4 month old male apoE4 mice compared to apoE3 and E2 mice (Sullivan et al., 2009
) (Riddell et al., 2008
). Another group found a trend towards lower apoE protein expression in the amygdala of female apoE4 TR mice (compared to apoE3 and apoE2 mice) however, no analysis of male TR mice was performed (Siegel et al., 2010
). Future studies are needed to determine the effects of gender and age on apoE protein levels.
carriers display signs of cognitive impairment by the second decade of life (Bourre et al., 1989
, Murthy et al., 2002
) (Ganguli et al., 2000
) (Scarmeas et al., 2005
) and on into middle age (Blair et al., 2005
) (Wishart et al., 2006
). Amygdalar volumes are also significantly reduced in probable AD patients based on MRI morphometry (Cuenod et al., 1993
) (Basso et al., 2006
). Determining when cognitive impairment begins in APOE4
carriers may reveal insight into the etiology of late onset AD and improve strategies for intervention. Here we provide the first electrophysiology studies in the human equivalent of “pre-adolescents” (i.e. pubescent 1 month old TR mice) which reveal an age and APOE
genotype effect on synaptic transmission in the amygdala. Ongoing and future studies in our lab aim to investigate the long-term impact of apoE isoforms on synaptic integrity in aged animals.