in situ hybridization techniques (Gazzaley et al., 1996b). Quantitative confocal microscopy was used to quantify alterations in immunofluorescence intensity levels of NR1 subunit proteins within neuronal cell bodies and dendrites of discrete hippocampal fields. In parallel, in situ hybridization was used to examine NR1 mRNA levels in corresponding hippocampal regions.
The data indicate that ERT in OVX rats significantly increases immunofluorescence intensity levels in comparison to nonsteroid-treated OVX rats within the cell bodies and dendrites of CA1 pyramidal cells and to a lesser extent within the granule cells of the dentate gyrus, without affecting CA3. In contrast, such alterations in immunofluorescence intensity occur without concomitant changes in mRNA hybridization levels. These data demonstrate a substantial degree of GluR plasticity in response to ERT over a fairly short period of time, since the ERT was initiated only one week after OVX, and the animals were sacrificed after only two days of ERT. These data are consistent with the estrogen-induced augmentation of NMDA receptor-mediated transmission by electrophysiological measurements (Woolley, 1999).
As compelling as these data are with respect to E-induced plasticity in the NMDA receptor, they are incomplete, in that we cannot comment on the effects on other related proteins (e.g., other NMDA receptor subunits, other GluRs.). In addition, we do not know how an increase in NR1 at the level of the soma and dendritic shafts translates into a change at the synapse. Moreover, these experiments were performed on young rats. Thus, while the receptor analyses on aged monkeys and estrogen-manipulated young rats suggest that both aging and endocrine status can alter NMDA receptors in a profound and circuit-dependent manner, the appropriate multidisciplinary analyses have yet to be carried out to determine if such alterations directly impact age-related memory decline. Studies of young and aged primates that are closely monitored both behaviorally and endocrinologically will need to be done to properly extend these studies. In addition, these studies will have to be extended to the ultrastructural level, in order to determine whether or not such dendritic shifts in NR1 are manifested at the synapse. Thus, while certain NMDA receptor-mediated hippocampal circuits are excellent targets for gene/circuit/behavioral links to be established in the context of age-related cognitive decline, most of the data needed to solidify such links are still missing.
While neuroanatomic datasets and behavioral datasets can be compared across experiments, it is most powerful when the neuroanatomic and cellular analyses are done in the same animals that have been behaviorally characterized. This has been particularly powerful in the hands of investigators that behaviorally screen aged animals so that the behaviorally impaired aged ani-