Nuclear and extranuclear estrogen receptor-alpha in the hippocampus

Estrogens are traditionally thought to act through a nuclear receptor-mediated mechanism to regulate gene transcription in target tissues. The hippocampus has been shown to be sensitive to estrogen; estrogen regulates GABAergic inhibition and spine density of dorsal CA1 pyramidal cells. However, very few neurons expressing nuclear estrogen receptors (ERs) have been detected in the hippocampus. I used double-label immunohistochemistry (Chapter 2) to quantify and map cells expressing nuclear ER-alpha (ERalpha) and/or cytoplasmic GABA in the hippocampus and determined that, in the dorsal CA1 region, nuclear ERalpha is expressed exclusively in inhibitory interneurons, but is limited to a small subset of those, while in the ventral hippocampus, ∼50-60% of pyramidal cells express nuclear ERalpha as well. Rapid estrogen effects observed in the hippocampal CA1 suggest that estrogen may act via extranuclear ERs. In order to determine a potential role of extranuclear ERalpha in the dorsal hippocampus, I used serial electron microscopy (Chapter 3) to create 3-D reconstructions of inhibitory axons innervating somata of CA1 pyramidal cells and discovered that only a portion of boutons on a given axon contain ERalpha-IR and in those, approximately 10% of presynaptic vesicles contain extranuclear ERalpha. Vesicles containing ERalpha-IR are always clustered together within a bouton and are located significantly closer to the synapse 24 hours after estrogen treatment. In order to assess the scope of extranuclear ERalpha expression, I used double-label immunofluorescence and confocal microscopy (Chapter 4) to determine that approximately 32% of GABAergic varicosities in the CA1 pyramidal cell layer contain ERalpha-IR. Furthermore, of the two neurochemically distinct subpopulations of basket cells in CA1, only axonal varicosities arising from cholecystokinin basket cells contained ERalpha-IR. Addition analysis also revealed that approximately 50% of ERalpha-IR puncta colocalized with NPY-IR. The presence of ERalpha near inhibitory synapses and the sensitivity of ERalpha-IR vesicle clusters to estrogen suggest that estrogen acts directly through non-nuclear ERalpha in a special subset of cholecystokinin boutons to regulate GABAergic inhibition of CA1 pyramidal cells. The results of these experiments increase our understanding of ERs in the hippocampus and lay the foundation for elucidating new mechanisms of estrogen action in the hippocampus.