Identification of dopaminergic neurons of the substantia nigra pars compacta as a target of manganese accumulation

Abstract

Manganese serves as a cofactor to a variety of proteins necessary for proper bodily development and function. However, an overabundance of Mn in the brain can result in manganism, a neurological condition resembling Parkinson's disease (PD). Bulk sample measurement techniques have identified the globus pallidus and thalamus as targets of Mn accumulation in the brain, however smaller structures/cells cannot be measured. Here, X-ray fluorescence microscopy determined the metal content and distribution in the substantia nigra (SN) of the rodent brain. In vivo retrograde labeling of dopaminergic cells (via FluoroGold™) of the SN pars compacta (SNc) subsequently allowed for XRF imaging of dopaminergic cells in situ at subcellular resolution. Chronic Mn exposure resulted in a significant Mn increase in both the SN pars reticulata (>163%) and the SNc (>170%) as compared to control; no other metal concentrations were significantly changed. Subcellular imaging of dopaminergic cells demonstrated that Mn is located adjacent to the nucleus. Measured intracellular manganese concentrations range between 40-200 μM; concentrations as low as 100 μM have been observed to cause cell death in cell cultures. Direct observation of Mn accumulation in the SNc could establish a biological basis for movement disorders associated with manganism, specifically Mn caused insult to the SNc. Accumulation of Mn in dopaminergic cells of the SNc may help clarify the relationship between Mn and the loss of motor skills associated with manganism.

Fig. 1

Tissue level XRF analysis of the SN. (A) Schematic of the coronal plane illustrating the substantia nigra (SN). The substantia nigra pars reticulata (SNr) separates the hippocampal formation (HPCf) and the substantia nigra pars compacta (SNc), which accumulates Mn. The red box corresponds to the regions featured in B. For clarity, the cerebral peduncle (cp) is also labeled. (B) XRF images of the SNr and the SNc, identifiable by the strong Fe and Mn signals respectively. The high Zn content to the lower left is due to the CA3 layer of the HPCf. Maximum scale for Mn in control is one-third that of the Mn treated sample for viewing purposes. (C) Tri-colored plots demonstrate the highest Mn accumulation adjacent to the SNr in both control and treated animals. Brain areas of SNr, SNc, caudal peduncle (cp), and HPCf are indicated on the plot. (D) Scatter plots of average metal concentration versus Mn for the SNc (dark tint) and SNr (light tint) for control (blue, n = 16 points) and Mn treated animals (red, n = 12 points). Gray lines indicate the best fit of the data weighted according to standard deviation of the individual data points (bars). Grayed data points identified as outliers and not considered for fitting purposes. Pearson correlation coefficient (r) and significance level (**p < 0.01) are displayed for metal/Mn of treated samples (Table S1, ESI‡). All numbers given are in μg g⁻¹.

Fig. 2

FG labeling of DA cells. (A) Diagram of a sagittal plane of the rodent brain with the approximate location of FG injection. The red arrow gives an approximate route of the nigra-neostriatum pathway. Injection into the CPu (B) results in uptake into axonal terminals and transport to DA cell bodies of the SNc (C). Scale bars represent a length of 100 μm (10 × objective). (D) Tyrosine hydroxylase IHC staining (green) reveals a more dense population of DA cells in the SNc than FG tracer (red), however all FG accumulating cells are positive for TH verified by two color image. Scale bars represent a length of 25 μm (40 × objective).

Fig. 3

Optical and XRF imaging of FG, Mn treated rat brain. (A) Brightfield (BF) image of the SNc (gray) with FG fluorescence image overlayed (yellow). Not all dopaminergic cells of the SNc are FG positive. (B) Fly scan XRF image of K and Zn that assist in co-localizing XRF imaging with BF/FG optical images. Dashed white box indicates an area measured at high resolution which contains two cells (C). Example of XRF image recorded at subcellular resolution (0.5 μm × 0.5 μm). A high Zn content was taken to indicate the location of the nucleus. All numbers given are in μg g⁻¹.