ificantly increase dendritic spine density compare to controls, suggesting that the ligand binding domain of ApoEr2 alone is not sufficient to induce spine formation. The extracellular and intracellular domains of ApoEr2 are important for dendritic spine formation Our previous findings demonstrated that ApoEr2 promotes dendritic spine formation. Therefore, we then examined which domains of ApoEr2 are essential to this process. To test this, we used deletion constructs of ApoEr2, as previously described . Primary hippocampal neurons were transfected with GFP and empty vector, GFP and ApoEr2 lacking the ligand binding domain, GFP and ApoEr2 lacking the ligand binding and EGF binding domains, GFP and ApoEr2 lacking the ligand binding, EGF binding and O-linked domains, and GFP and full-length ApoEr2, and spine ApoEr2 deficient mice have decreased spine density at 1 month of age To determine whether ApoEr2 has a similar effect on dendritic spine formation in vivo, we analyzed pyramidal neurons in cortical layers II/III using the rapid Golgi impregnation method in ApoEr2 knockout mice and wild-type littermates at 1 month old. Because spine density may vary within both the dendritic field of a single neuron and between different neurons, the spines of the pyramidal neurons were counted as apical oblique, basal shaft, and total dendrites. We found that ApoEr2 knockout mice had significantly reduced dendritic spine number on AO, BS, and total dendrites compared with wild-type littermates at 1 month old. 5 February 2011 | Volume 6 | Issue 2 | e17203 The Effect of ApoEr2 on Dendritic Spine Formation Next, we examined the morphology of dendritic spines by measuring spine width and length in ApoEr2 knockout mice and wild-type littermates at 1 month old. The cumulative distributions revealed no significant differences in spine width between ApoEr2 knockout mice and wild-type mice. However, we observed a significant shift in 1 month old ApoEr2 knockout mice to shorter spines compared to wild-type mice in the AO, but not BS dendrites. We further examined whether the 
effects of ApoEr2 on dendritic spine density was age-dependent. To test this, we conducted Golgi staining on ApoEr2 knockout mice and wild-type littermates at 1 year of age. Interestingly, we observed no significant differences in dendritic spine densities between ApoEr2 knockout mice and wild-type littermates in cortical layers II/III at 1 year old. This data suggests the existence of compensatory mechanisms between one month of age and one year of age in ApoEr2 knockout mice. We also conducted morphological analysis in 1 year old ApoEr2 knockout mice and wild-type littermates. The cumulative distributions revealed a significant shift in ApoEr2 knockout mice to shorter spines compared to wild-type mice in the February 2011 | Volume 6 | Issue 2 | e17203 The Effect of ApoEr2 on Dendritic Spine Formation AO, but not BS dendrites, consistent with our 1 month of age studies. ApoEr2 increased clustering of synaptic proteins Because our data indicate that ApoEr2 plays an important role in promoting synapse and dendritic spine numbers, we examined whether ApoEr2 affects clustering of synaptic proteins. For these experiments, primary hippocampal neurons were infected with ApoEr2 sindbis virus or GFP for 20 hours and 216450-65-6 intensity or puncta number of PSD-95 and synaptophysin were measured. We found that ApoEr2 significantly increased PSD-95 intensity throughout the cultures by 38% compared to GFP infect