Human Studies Interrogating the Hippocampal Circuit in AD and Aging

By quantifying cell loss in postmortem tissue of AD patients, studies have suggested that either the entorhinal cortex or the CA1 subfield is the hippocampal subregion most vulnerable to AD (Braak and Braak, 1996; Fukutani, Cairns et al., 2000; Price, Ko et al., 2001; Shoghi-Jadid, Small et al., 2002; Giannakopoulos, Herrmann et al., 2003; Schonheit, Zarski et al., 2004). In many of these studies, the entorhinal cortex and the CA1 subfield were not assessed simultaneously, accounting in part for the reported inconsistencies. More generally, however, isolating the hippocampal subregion most vulnerable to AD is difficult relying on postmortem studies alone. Not only are postmortem series biased against the earliest and most discriminatory stages of disease, but these studies cannot assess the cell-sickness stage of AD (Selkoe, 2002).

As discussed above, variants of fMRI sensitive to basal correlates of neuronal function are well suited to aid in resolving this debate. In one study, an MRI measure sensitive to basal levels of deoxyhemoglobin was used to assess the hippocampal subregions in patients with AD dementia compared to age-matched controls (Small, Nava et al., 2000). This MRI measure has proven capable to detect cell sickness in individual hippocampal sub-regions. Univariate analysis revealed that normalized signal intensity was reduced in all hippocampal sub-regions in patients compared to controls. Nevertheless, when the hippocampus was analyzed as a circuit— namely, using a multivariate model to analyze signals from all hippocampal subregions simultaneously—the entorhinal cortex was found to be the primary site of dysfunction in AD (Small, Nava et al., 2000).

This study suggests that the entorhinal cortex, not the CA1 subfield, is the hippocampal subregion most vulnerable to AD—agreeing with some, though not all, postmortem studies. Nevertheless, the patients assessed in this study already had full-blown dementia, indicating that they had already progressed from the cell-sickness to the cell-death stage of AD. Furthermore, this study does not inform us about the hippocampal subregions most vulnerable to normal aging.

Both of these issues were addressed in a second study, in which 70 subjects across the age span—from 20 to 88 years of age—were imaged with the same MRI measure (Small, Tsai et al., 2002). Importantly, all subjects were healthy. The older age groups in particular were carefully screened against any evidence of dementia. The starting assumption made in this study was that some of the older subjects were in the earliest stage of AD and some subjects were aging normally. The question was how to make this distinction. Remember, there is no independent indicator to determine who had early AD or not. This is true even if the hippocampal formation of all subjects could be examined postmortem, because, as mentioned, the earliest stages of AD may be invisible to the microscope. Instead, formal parametric criteria were used to distinguish a ''pathological pattern'' of decline (i.e., related to Alzheimer's disease) versus a ''normal pattern'' of decline. Specifically, since the effect of normal aging on the brain is, by definition, a stochastic process, the variance of signal intensity among an older age group should be equal to the variance among a younger age group, although a shift in the mean is expected. In contrast, since it is a disease, AD should affect a subgroup within an older age group, which should significantly broaden the variance of signal intensity compared to a younger age group.

Applying this and other criteria, the results of this study showed that age-related changes in the entorhinal cortex fulfilled criteria for pathological decline; in contrast, age-related changes in the dentate gyrus, and to a lesser extent in the subiculum, fulfilled criteria for normal aging (Small, Tsai et al., 2002). These findings not only confirm but also extend the results of the previous study. First, the entorhinal cortex indeed appears to be the hippocampal subregion most vulnerable to AD, even during the early cell-sickness stage. Second, these findings provided the first evidence that the dentate gyrus might be the hippocampal subregion most vulnerable to normal aging.

This study had a number of limitations. First, despite the strict criteria, independent verification of which older subjects did or did not have early AD was not possible. Second, although the MRI measure used is sensitive to basal deoxyhemoglobin levels, these images are also sensitive to other, nonmetabolic, tissue constituents that are potential confounds (Small, Wu et al., 2000; Small, 2003).

These potential limitations were addressed in a third study. First, a cohort of aging individuals was needed that indisputably were free of AD. Because this cohort is difficult, or even impossible, to identify in human subjects, we turned to aging nonhuman primates instead. Like all mammals, monkeys develop age-related hippo-campal dysfunction, yet they do not develop the known molecular or histological hallmarks of AD. Second, because of the stated limitations of imaging techniques sensitive to deoxyhemoglobin content, we relied on MRI to generate regional measures of cerebral blood volume (CBV). Previous studies have established that CBV is a hemodynamic variable tightly correlated with brain metabolism, capable of detecting brain dysfunction in the hippocampus and other brain regions (Gonzalez, Fischman et al., 1995; Harris, Lewis et al., 1998; Wu, Bruening et al., 1999; Bozzao, Floris et al., 2001).

In the third study, the hippocampal subregions of 14 rhesus monkeys were imaged across the age span from 7 to 31 years of age (Small, Chawla et al., 2004). In a remarkable parallel to the previous human study, age-related decline in CBV was observed only in the dentate gyrus, and to a lesser extent the subiculum. Notably, CBV measured from the entorhinal cortex and the CA1 subregion remained stable across the life span. Indeed, when all subregions were analyzed simulta-neously—in accordance with the circuit organization of the hippocampus—the dentate gyrus was the primary subregion that declined with age. Furthermore, since all monkeys were assessed cognitively, we found that a decline in dentate gyrus CBV was the only subregion that correlated with a decline in memory performance (Small, Chawla et al., 2004).

Despite the reliance on CBV to investigate aging monkeys, this third study also had a number of limitations. The first limitation applies to all functional imaging: As derived from Fick's principle (Small, 2004), all hemodynamic variables—deoxyhemoglobin, CBV, or cerebral blood flow (CBF)—are correlates of oxygen metabolism; nevertheless, they are only indirect correlates. The possibility always exists that these measures are confounded by changes in vascular physiology, and not underlying neuronal physiology. Thus, we cannot exclude the possibility that there is something unique to the vascular system within the dentate gyrus that caused shrinkage of CBV, independent of dentate gyrus physiologic dysfunction. The second limitation of the monkey MRI study has to do with the cellular complexity of any brain subregion, including the dentate gyrus. Although the granule cells are the primary neurons of the dentate gyrus, the dentate gyrus contains other types of neurons as well as glial cells. Even if the CBV measure does reflect underlying cellular function, MRI cannot be relied on to isolate the cells that govern this observed effect.

A fourth study was designed to address these concerns. Here, in vitro imaging was used, directly visualizing correlates of neuronal physiology. Aging rats were investigated, who like humans and monkeys develop age-related hippocampal dysfunction. Immunocyto-chemisty was used to visualize the behaviorally-induced expression of Arc in the hippocampal subregions of aging rats. Arc is an immediate early gene whose expression has been shown to correlate with spike activity and with long-term plasticity in hippocampal neurons (Guzowski, McNaughton et al., 1999; Guzowski, Lyford et al., 2000; Guzowski, Setlow et al., 2001). Rats of different ages were allowed to explore a novel place and sacrificed and processed for Arc staining. Arc expression was quantified in the granule cells of the dentate gyrus and in the pyramidal neurons of the CA1 and CA3 subregions. The dentate gyrus was the only hippocampal subregion whose neurons were found to have a significant age-related decline in Arc expression (Small, Chawla et al., 2004). Thus, this study confirms and extends the prior studies, showing that it is in fact neuronal, not vascular, physiology that underlies the aging effect. Moreover, this study established that aging primarily targets the granule cells of the dentate gyrus.

To summarize, by using different imaging techniques across three different mammalian species (Small, Tsai et al., 2002; Small, Pierce et al., 2003), a consensus has emerged from these complementary studies: That the dentate gyrus is the hippocampal subregion differentially vulnerable to the aging process, and that it is the primary subregion underlying age-related hippocampal dysfunction and memory decline.

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