Stereologic estimates of total spinophilin-immunoreactive spine number in area 9 and the CA1 field: relationship with the progression of Alzheimer's disease

Abstract

The loss of presynaptic markers is thought to represent a strong pathologic correlate of cognitive decline in Alzheimer's disease (AD). Spinophilin is a postsynaptic marker mainly located to the heads of dendritic spines. We assessed total numbers of spinophilin-immunoreactive puncta in the CA1 and CA3 fields of hippocampus and area 9 in 18 elderly individuals with various degrees of cognitive decline. The decrease in spinophilin-immunoreactivity was significantly related to both Braak neurofibrillary tangle (NFT) staging and clinical severity but not A beta deposition staging. The total number of spinophilin-immunoreactive puncta in CA1 field and area 9 were significantly related to MMSE scores and predicted 23.5 and 61.9% of its variability. The relationship between total number of spinophilin-immunoreactive puncta in CA1 field and MMSE scores did not persist when adjusting for Braak NFT staging. In contrast, the total number of spinophilin-immunoreactive puncta in area 9 was still significantly related to the cognitive outcome explaining an extra 9.6% of MMSE and 25.6% of the Clinical Dementia Rating scores variability. Our data suggest that neocortical dendritic spine loss is an independent parameter to consider in AD clinicopathologic correlations.

Figure 1

Photomicrographs of sections through the hippocampus (A) and area 9 (B). A: Coronal section at the level of hippocampus showing the contours of CA1 (red) and CA3 (blue). B: Photomicrograph demonstrating spinophilin labeling in area 9 (arrows mark the boundaries). There is intense spinophilin immunoreactivity in all of the cortical layers, but none in the white matter. Scale bars = 1 mm.

Figure 2

Photomicrograph showing spinophilin-immunoreactive puncta at 100x magnification (A–F). Spines not visualized at their optimal focal plane appear blurred. (A) Spinophilin immunolabeling in CA1 section (CDR 0) at the magnification used for stereologic analysis. (B) Spinophilin-immunolabeled CA1 section (CDR 3). An unbiased counting frame is shown. The red border of the frame and its extensions represent the exclusion line and the green border represents the inclusion line. Spines were counted when they came into focus within the height of the optical disector and within the counting frame when moving the focal plane continuously through the section. A spine was counted if it was entirely within the counting frame or partially within it without touching or intersecting the exclusion line when in focus. (C, D) High magnification images of spinophilin immunoreactivity in a CA3 section at CDR 0 and 3 respectively. (E, F) High magnification images of spinophilin immunoreactivity in area 9 at CDR 0 and 2 respectively. Scale bar = 20 µm.

Figure 3

Regression lines with 95% confidence intervals of spinophilin-immunoreactive puncta (in billions) versus Braak NFT staging, Aβ deposition staging, MMSE, and CDR scores in the CA1 field and area 9. Note the significant negative association between Braak NFT staging and total spinophilin-immunoreactive puncta in both the CA1 field and area 9. Note also the negative relationship between clinical indices and total spinophilin-immunoreactive puncta in the same areas. See text for details.