Sein Cho
Johns Hopkins University
The hippocampus has been central in the study of memory formation and consolidation. The functional specialization and selectivity that subregions of the hippocampus demonstrate has been critical in studying how they respond and change to aging. With memory impairment being a high-stake concern in the aging population, various studies have looked at how the functional properties of hippocampal circuits change. Using glutamic acid decarboxylase-67 (GAD67), somatostatin, and neuropeptide Y immunocytochemical markers, previous research in the lab has found that age-related decreases in GAD67- and somatostatin-immunoreactive neuron number beyond the hilus were regionally selective, entirely sparing the CA1 area. (Spiegel et al., 2013) Using male Long-Evans rats with a spectrum of individual differences in hippocampal-dependent memory, it was found that the alterations of interneurons specifically related to cognitive outcomes of aging were observed exclusively in the hilus of the dentate gyrus (DG). The study provided insight into the vulnerability of hippocampal interneurons to normal aging and suggested that a specific subpopulation in the hilus is coupled with age-related memory impairment. The current project aims to replicate the work with female rats, which would potentially further support the conclusion that the total number of hilar interneurons is correlated with memory performance among aged rats. It would also be worth noting any sex-specific differences in the expression of immunopositive interneurons.
Introduction
The hippocampus, widely regarded as the center of the episodic memory network, is known for its important function in learning as well as the consolidation of memory. It is distinguished by its tri-synaptic pathway which originates at the entorhinal cortex, projects to the dentate gyrus (DG), and further on to area CA3 and CA1 (Alkadhi, 2019). Each of these areas perform distinct functions with their unique cellular structure. Such regional and cellular specialization of the hippocampus has led to the investigation of how it changes in response to aging. Aging is usually accompanied by memory loss. Regions in the hippocampus have been shown to be especially prone to memory impairment from aging, and changes in the hippocampal circuit have been identified. The dentate gyrus (DG) and CA3 subregions have shown increased activation with age-related memory loss or amnestic mild cognitive impairment (Yassa et al., 2010, 2011).
A decline in hippocampal interneuron integrity is associated with normal brain aging (Bartsch, 2015). However, equally important is to initially identify the cognitive status of aged subjects being used in the study to more clearly define the relationship between interneuron integrity and cognitive decline. This was done by a previous study in the lab, which tested the relationship between interneuron network integrity and age-related memory impairment. Using outbred, aged, male Long-Evans rats with a wide spectrum of differences in hippocampal-dependent memory, immunocytochemical markers GAD67, somatostatin and neuropeptide Y were used to quantify numbers of hippocampal interneurons. A decline GAD67- and somatostatin-positive interneuron numbers were found across multiple areas of the hippocampus, with the hilus of the dentate gyrus demonstrating alterations specifically related to cognitive outcome of aging. In the current study, parvalbumin was used as an immunocytochemical marker in place of neuropeptide Y. The primary focus of the current work is to determine whether age- and cognition-related decrease in interneuron subpopulations is also observed in female rats.
Methods
Aged male and female Long-Evans rats were used for the study. All rats were individually housed and maintained on a 12-hour light/dark cycle and were determined to be healthy and free of pathogenesis and disability. The water maze task was used to behaviorally characterize the rats for spatial memory. Approximately 1 month after behavioral characterization, the animalswere anesthetized with isoflurane and perfused for tissue preparation and collection.
Following antibody characterization with relevant host species and antigens, selected interneuron populations in the hippocampus were stained with immunocytochemical markers, including GAD67, somatostatin and parvalbumin. I specifically focused on working with GAD67 immunohistochemistry to help quantify the number of interneurons within certain subfields of the hippocampus. The histological sections were processed according to an established protocol (Spiegel et al.,, 2013; Haberman et al., 2011). After primary and secondary antibody incubationalong with washes in PBS, tissue sections were mounted onto gelatin-coated slides and dried, dehydrated with increasing ethanol concentrations, cleared in xylene, and coverslipped using DPX mounting media. Different trials were carried out before finalizing the concentration of the relevant stain (thionin, neutral red, cresyl violet stain) that resulted in the best visualization of the cell body. The staining protocol was strictly followed regarding the amount of time the slides were in each bucket, usually starting out with decreasing concentrations of ethanol followed by shorter dips in double-distilled water, the relevant staining media, and finishing off with increasing concentration of ethanol.
The sequence of slides and regions of interest (ROIs) were defined according to the Swanson rat brain atlas (1992). 8 sections of tissue sectioned on a freezing microtome in the coronal plane were identified by different levels. For ROI identification, levels 28 to 35 were identified sequentially, accounting for individual differences across the animals. Using overlays within the ImageJ software, the slices were annotated for area and magnification. For interneuron quantification within each subfield, the number of interneurons within each ROI is currently being counted. PARV analyses are being conducted for the DG and SOM analyses are beingconducted for the hilus and area CA3. GAD-67 analyses for interneuron counts are in the process for areas of the hilus, CA3 and CA1. Analyses are being conducted blind with regard to animal age and cognitive status.
Figure 1. Tissue sections mounted onto slides
Figure 2. ROIs (hilus of dentate gyrus, CA3) identified for animal 2010_21 (slice #6)
Results/Discussion
With the results of total cell counts for each immunocytochemical marker, statistical analyses will be completed within young and aged rates for each hippocampal region and sublayer. For example, analyzing GAD67-postive neuron number across the hilus, CA3 and CA1 subfields will allow us to draw a conclusion about whether there is an age-related decrease in the number of immunopositive neurons. The results of the current study is expected to support and potentially investigate sex-specific assessments between hippocampal GABAergic interneurons in aged rats with cognitive impairment, relative to those aged and young with intact memory.
References
Alkadhi, K.A. Cellular and Molecular Differences Between Area CA1 and the Dentate Gyrus of the Hippocampus. Mol Neurobiol 56, 6566–6580 (2019). https://doi.org/10.1007/s12035-019-1541-2
Bartsch, T., & Wulff, P. (2015). The hippocampus in aging and disease: From plasticity to vulnerability. Neuroscience, 309, 1–16. https://doi.org/10.1016/j.neuroscience.2015.07.084
Haberman, R. P., Colantuoni, C., Stocker, A. M., Schmidt, A. C., Pedersen, J. T., & Gallagher, M. (2011). Prominent hippocampal CA3 gene expression profile in neurocognitive aging. Neurobiology of aging, 32(9), 1678–1692. https://doi.org/10.1016/j.neurobiolaging.2009.10.005
Spiegel, A. M., Koh, M. T., Vogt, N. M., Rapp, P. R., & Gallagher, M. (2013). Hilar interneuron vulnerability distinguishes aged rats with memory impairment. The Journal of comparative neurology, 521(15), 3508–3523. https://doi.org/10.1002/cne.23367
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