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Increasing evidence suggests that metal dyshomeostasis plays an important role in human neurodegenerative diseases. Although distinctive metal distributions are described for mature hippocampus and cortex, much less is known about metal levels and intracellular distribution in individual hippocampal neuronal somata. To solve this problem, we conducted quantitative metal analyses utilizing synchrotron radiation X-Ray fluorescence on frozen hydrated primary cultured neurons derived from rat embryonic cortex (CTX) and two regions of the hippocampus: dentate gyrus (DG) and CA1. Comparing average metal contents showed that the most abundant metals were calcium, iron, and zinc, whereas metals such as copper and manganese were less than 10% of zinc. Average metal contents were generally similar when compared across neurons cultured from CTX, DG, and CA1, except for manganese that was larger in CA1. However, each metal showed a characteristic spatial distribution in individual neuronal somata. Zinc was uniformly distributed throughout the cytosol, with no evidence for the existence of previously identified zinc-enriched organelles, zincosomes. Calcium showed a peri-nuclear distribution consistent with accumulation in endoplasmic reticulum and/or mitochondria. Iron showed 2-3 distinct highly concentrated puncta only in peri-nuclear locations. Notwithstanding the small sample size, these analyses demonstrate that primary cultured neurons show characteristic metal signatures. The iron puncta probably represent iron-accumulating organelles, siderosomes. Thus, the metal distributions observed in mature brain structures are likely the result of both intrinsic neuronal factors that control cellular metal content and extrinsic factors related to the synaptic organization, function, and contacts formed and maintained in each region.

Small alterations in the level of extracellular H + can profoundly alter neuronal activity throughout the nervous system. In this study, self-referencing H + -selective microelectrodes were used to examine extracellular H + fluxes from individual astrocytes. Activation of astrocytes cultured from mouse hippocampus and rat cortex with extracellular ATP produced a pronounced increase in extracellular H + flux. The ATP-elicited increase in H + flux appeared to be independent of bicarbonate transport, as ATP increased H + flux regardless of whether the primary extracellular pH buffer was 26 mM bicarbonate or 1 mM HEPES, and persisted when atmospheric levels of CO 2 were replaced by oxygen. Adenosine failed to elicit any change in extracellular H + fluxes, and ATP-mediated increases in H + flux were inhibited by the P2 inhibitors suramin and PPADS suggesting direct activation of ATP receptors. Extracellular ATP also induced an intracellular rise in calcium in cultured astrocytes, and ATP-induced rises in both calcium and H + efflux were significantly attenuated when calcium re-loading into the endoplasmic reticulum was inhibited by thapsigargin. Replacement of extracellular sodium with choline did not significantly reduce the size of the ATP-induced increases in H + flux, and the increases in H + flux were not significantly affected by addition of EIPA, suggesting little involvement of Na + /H + exchangers in ATP-elicited increases in H + flux. Given the high sensitivity of voltage-sensitive calcium channels on neurons to small changes in levels of free H + , we hypothesize that the ATP-mediated extrusion of H + from astrocytes may play a key role in regulating signaling at synapses within the nervous system.