Quantitative analysis of zinc in rat hippocampal mossy fibers by nuclear microscopy

Zinc (Zn) is involved in regulating mental and motor functions of the brain. Previous approaches have determined Zn content in the brain using semi-quantitative histological methods. We present here an alternative approach to map and quantify Zn levels in the synapses from mossy fibers to CA3 region of the hippocampus. Based on the use of nuclear microscopy, which is a combination of imaging and analysis techniques encompassing scanning transmission ion microscopy (STIM), Rutherford backscattering spectrometry (RBS), and particle induced X-ray emission (PIXE), it enables quantitative elemental mapping down to the parts per million (μg/g dry weight) levels of zinc in rat hippocampal mossy fibers. Our results indicate a laminar-specific Zn concentration of 240 ± 9 μM in wet weight level (135 ± 5 μg/g dry weight) in the stratum lucidum (SL) compared to 144 ± 6 μM in wet weight level (81 ± 3 μg/g dry weight) in the stratum pyramidale (SP) and 78 ± 10 μM in wet weight level (44 ± 5 μg/g dry weight) in the stratum oriens (SO) of the hippocampus. The mossy fibers terminals in CA3 are mainly located in the SL. Hence the Zn concentration is suggested to be within this axonal presynaptic terminal system.

► Free Zn is highly concentrated in the mossy fiber terminals in the hippocampus.
► Regulation of Zn concentration is critical for brain health and function.
► We mapped and quantified Zn levels in three layers of CA3 using nuclear microscopy.
► Total Zn in SL, SP and SO layer of CA3 was accurately calculated at μg/g dry weight.