Visualizing Metal Content and Intracellular Distribution in Primary Hippocampal Neurons with Synchrotron X-Ray Fluorescence

Abstract

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.

Fig 1. 2-D scans and line scan data derived from SRXRF analysis of frozen hydrated primary neurons cultured from the dentate gyrus.

The relative intensity rainbow scale used is shown below the potassium scan (A)–red is highest, black is lowest– μg/cm². Image size calibration is shown as a black bar, below the potassium scan. (A) Potassium, (B) sulfur, (C) zinc, (D) calcium, (E) iron, and (F) manganese. (E1) Shows a blow-up of the region outlined in the white box in E illustrating iron puncta and N represents the approximate location of the nucleus. In E1 the scale is changed to allow better visualization of the iron distribution in the soma. Using this scale, the iron puncta are over-saturated (see white arrows pointing to red puncta). Shown in the potassium 2-D scan (A) is a red arrow that illustrates the location and direction of the line scan that produced the plots: (G) zinc and potassium; (H) calcium and potassium; (I) iron and potassium; (J) iron, calcium and zinc. The maximum analytical variance calculated for these data: K = 0.13 μg/cm², Ca = 0.03 μg/cm², Fe = 0.01 μg/cm², Zn = 0.005 μg/cm². Note that the 2-D scans and line scan data are not sample background corrected. Sample backgrounds for this SiN window were calculated as (μg/cm²): potassium = 0.194; sulfur = 0.034; zinc = 0.005; calcium = 0.02; iron = 0.009; and manganese = 0.004. (K) Light microscopic image of dentate gyrus neurons prior to flash freezing. Images were taken using 20x NA 0.3 Zeiss LD A-Plan Hoffman Modulation Contrast lens and Zeiss Axiovert S100 microscope controlled by Zeiss Axiovision v. 4.9.1.0 software. Bar equals 50 μm.

Fig 2. Schematic of an individual neuron soma and how it correlates to line scan fluorescence data.

The red arrow depicts the direction of the scan as shown in Fig 1A. The line scan data shown are taken from Fig 1I. A single iron punctum is shown in a peri-nuclear region within the soma.

Fig 3. Average metal contents of neuronal soma from hippocampal and cortical primary neurons.

Regions of interest (ROI) were manually drawn around neuronal soma observed in 2-D SRXRF scans using MAPS software (see Methods) and metal contents are expressed as the average value for each soma (ng/cm²) with sample background subtracted. The data are displayed as scatter plots (horizontal bar represents the mean of all measurements for that experimental condition): (A) zinc, (B) iron, (C) manganese, and (D) copper. Each filled circle represents data obtained from one neuron; an ROI manually drawn around its soma. For each cell type, data were collected from a single SiN window. *—Comparing calculated means for each metal across cell types, only manganese CA1 was found to be significantly greater than manganese dentate gyrus–p < 0.05, Kruskal-Wallis test with Dunn's Multiple Comparison Test.

Fig 4. Average iron content in regions of interest (ROI) manually drawn around iron puncta (Fe p., identified in 2-D SRXRF scans; see Fig 1E1 for examples).

Metal contents are expressed for each ROI as an average value (ng/cm²) with sample background subtracted. The data are displayed as scatter plots (horizontal bar represents the mean of all measurements for that experimental condition), filled circles represent individual iron puncta analyzed for each cell type. Data for iron puncta were collected from 5 DG cells, 7 CA1 cells, and 4 cortical cells. For each cell type, data were collected from a single SiN window. *—Comparing calculated means for each cell type, Kruskal-Wallis test with Dunn's Multiple Comparison Test, p < 0.05 for iron puncta (Fe p.) dentate gyrus versus CA1 neurons.