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Ion exchanger in the brain: Quantitative analysis of perineuronally fixed anionic binding sites suggests diffusion barriers with ion sorting properties.

Morawski M, Reinert T, Meyer-Klaucke W, Wagner FE, Tröger W, Reinert A, Jäger C, Brückner G, Arendt T - Sci Rep (2015)

Bottom Line: For the first time, we can provide quantitative data on the distribution and net amount of pericellularly fixed charge-densities, which, determined at 0.4-0.5 M, is much higher than previously assumed.PNs, thus, represent an immobilized ion exchanger with ion sorting properties high enough to partition mobile ions in accord with Donnan-equilibrium.We propose that fixed charge-densities in the brain are involved in regulating ion mobility, the volume fraction of extracellular space and the viscosity of matrix components.

View Article: PubMed Central - PubMed

Affiliation: Paul Flechsig Institute for Brain Research, University of Leipzig, Liebigstrasse 19, D04103 Leipzig, Germany.

ABSTRACT
Perineuronal nets (PNs) are a specialized form of brain extracellular matrix, consisting of negatively charged glycosaminoglycans, glycoproteins and proteoglycans in the direct microenvironment of neurons. Still, locally immobilized charges in the tissue have not been accessible so far to direct observations and quantifications. Here, we present a new approach to visualize and quantify fixed charge-densities on brain slices using a focused proton-beam microprobe in combination with ionic metallic probes. For the first time, we can provide quantitative data on the distribution and net amount of pericellularly fixed charge-densities, which, determined at 0.4-0.5 M, is much higher than previously assumed. PNs, thus, represent an immobilized ion exchanger with ion sorting properties high enough to partition mobile ions in accord with Donnan-equilibrium. We propose that fixed charge-densities in the brain are involved in regulating ion mobility, the volume fraction of extracellular space and the viscosity of matrix components.

No MeSH data available.


Light micrograph (LM) and quantitative PIXE elemental images of two PN-ensheathed neurons in rat brainstem (7 μm thick section, PNs visualized using WFA-binding enhanced by DAB-Ni staining (grey-black pigment)).(Ni) In the nickel map, PNs are visible due to the Ni-accumulation after immunohistochemical staining. (P) The phosphorus map mainly represents the distribution of the phosphate rich RNA and DNA, i.e. the Nissl substance in the neuronal cytoplasm and the nucleolus, but also glia cell nuclei. (S) The sulphur distribution reflects the extracellular matrix. Due to the sulfate rich chondroitin components of the PN the concentration is higher at the PN. (Fe) The iron map shows diffuse cytosolic and nuclear distribution and a prominent signal over the nucleolus. (Fe-P-Ni) In the three-element image of phosphorus (green), nickel (blue) and iron (red), iron can clearly be allocated to subcellular compartments delineated by the phosphorus image. Elemental profiles are given for the traverse through the PN ensheathed neuron. Scale bar: 20 μm, top concentration at the color scale in mM.
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f1: Light micrograph (LM) and quantitative PIXE elemental images of two PN-ensheathed neurons in rat brainstem (7 μm thick section, PNs visualized using WFA-binding enhanced by DAB-Ni staining (grey-black pigment)).(Ni) In the nickel map, PNs are visible due to the Ni-accumulation after immunohistochemical staining. (P) The phosphorus map mainly represents the distribution of the phosphate rich RNA and DNA, i.e. the Nissl substance in the neuronal cytoplasm and the nucleolus, but also glia cell nuclei. (S) The sulphur distribution reflects the extracellular matrix. Due to the sulfate rich chondroitin components of the PN the concentration is higher at the PN. (Fe) The iron map shows diffuse cytosolic and nuclear distribution and a prominent signal over the nucleolus. (Fe-P-Ni) In the three-element image of phosphorus (green), nickel (blue) and iron (red), iron can clearly be allocated to subcellular compartments delineated by the phosphorus image. Elemental profiles are given for the traverse through the PN ensheathed neuron. Scale bar: 20 μm, top concentration at the color scale in mM.

Mentions: In order to analyze the fixed negative charge density in the neuronal microenvironment and PNs with an iron probe by μPIXE, the PN must be identified. Therefore, we applied an approach recently developed in our lab18 based on the application of antibodies tagged with an ultra-pure metallic label (e.g. Ni, Co, Cd, Ag, or Au), which combines immunohistochemistry and elemental imaging using μPIXE. Typical quantitative elemental images of brain sections obtained with this approach are shown in Fig. 1. PNs are identified in the nickel image. It shows the nickel accumulation due to the selective WFA-DAB-Ni-binding. The elemental profiles shown in Fig. 1 and control measurements (Supplementary Figure S1) verify that the Ni-enhancement of the histochemical staining does not affect the elemental imaging or the quantitative analysis of iron or other elements. The iron and nickel distributions are uncorrelated. Thus, an alteration of the iron concentration by the nickel-DAB-staining can be excluded.


Ion exchanger in the brain: Quantitative analysis of perineuronally fixed anionic binding sites suggests diffusion barriers with ion sorting properties.

Morawski M, Reinert T, Meyer-Klaucke W, Wagner FE, Tröger W, Reinert A, Jäger C, Brückner G, Arendt T - Sci Rep (2015)

Light micrograph (LM) and quantitative PIXE elemental images of two PN-ensheathed neurons in rat brainstem (7 μm thick section, PNs visualized using WFA-binding enhanced by DAB-Ni staining (grey-black pigment)).(Ni) In the nickel map, PNs are visible due to the Ni-accumulation after immunohistochemical staining. (P) The phosphorus map mainly represents the distribution of the phosphate rich RNA and DNA, i.e. the Nissl substance in the neuronal cytoplasm and the nucleolus, but also glia cell nuclei. (S) The sulphur distribution reflects the extracellular matrix. Due to the sulfate rich chondroitin components of the PN the concentration is higher at the PN. (Fe) The iron map shows diffuse cytosolic and nuclear distribution and a prominent signal over the nucleolus. (Fe-P-Ni) In the three-element image of phosphorus (green), nickel (blue) and iron (red), iron can clearly be allocated to subcellular compartments delineated by the phosphorus image. Elemental profiles are given for the traverse through the PN ensheathed neuron. Scale bar: 20 μm, top concentration at the color scale in mM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664884&req=5

f1: Light micrograph (LM) and quantitative PIXE elemental images of two PN-ensheathed neurons in rat brainstem (7 μm thick section, PNs visualized using WFA-binding enhanced by DAB-Ni staining (grey-black pigment)).(Ni) In the nickel map, PNs are visible due to the Ni-accumulation after immunohistochemical staining. (P) The phosphorus map mainly represents the distribution of the phosphate rich RNA and DNA, i.e. the Nissl substance in the neuronal cytoplasm and the nucleolus, but also glia cell nuclei. (S) The sulphur distribution reflects the extracellular matrix. Due to the sulfate rich chondroitin components of the PN the concentration is higher at the PN. (Fe) The iron map shows diffuse cytosolic and nuclear distribution and a prominent signal over the nucleolus. (Fe-P-Ni) In the three-element image of phosphorus (green), nickel (blue) and iron (red), iron can clearly be allocated to subcellular compartments delineated by the phosphorus image. Elemental profiles are given for the traverse through the PN ensheathed neuron. Scale bar: 20 μm, top concentration at the color scale in mM.
Mentions: In order to analyze the fixed negative charge density in the neuronal microenvironment and PNs with an iron probe by μPIXE, the PN must be identified. Therefore, we applied an approach recently developed in our lab18 based on the application of antibodies tagged with an ultra-pure metallic label (e.g. Ni, Co, Cd, Ag, or Au), which combines immunohistochemistry and elemental imaging using μPIXE. Typical quantitative elemental images of brain sections obtained with this approach are shown in Fig. 1. PNs are identified in the nickel image. It shows the nickel accumulation due to the selective WFA-DAB-Ni-binding. The elemental profiles shown in Fig. 1 and control measurements (Supplementary Figure S1) verify that the Ni-enhancement of the histochemical staining does not affect the elemental imaging or the quantitative analysis of iron or other elements. The iron and nickel distributions are uncorrelated. Thus, an alteration of the iron concentration by the nickel-DAB-staining can be excluded.

Bottom Line: For the first time, we can provide quantitative data on the distribution and net amount of pericellularly fixed charge-densities, which, determined at 0.4-0.5 M, is much higher than previously assumed.PNs, thus, represent an immobilized ion exchanger with ion sorting properties high enough to partition mobile ions in accord with Donnan-equilibrium.We propose that fixed charge-densities in the brain are involved in regulating ion mobility, the volume fraction of extracellular space and the viscosity of matrix components.

View Article: PubMed Central - PubMed

Affiliation: Paul Flechsig Institute for Brain Research, University of Leipzig, Liebigstrasse 19, D04103 Leipzig, Germany.

ABSTRACT
Perineuronal nets (PNs) are a specialized form of brain extracellular matrix, consisting of negatively charged glycosaminoglycans, glycoproteins and proteoglycans in the direct microenvironment of neurons. Still, locally immobilized charges in the tissue have not been accessible so far to direct observations and quantifications. Here, we present a new approach to visualize and quantify fixed charge-densities on brain slices using a focused proton-beam microprobe in combination with ionic metallic probes. For the first time, we can provide quantitative data on the distribution and net amount of pericellularly fixed charge-densities, which, determined at 0.4-0.5 M, is much higher than previously assumed. PNs, thus, represent an immobilized ion exchanger with ion sorting properties high enough to partition mobile ions in accord with Donnan-equilibrium. We propose that fixed charge-densities in the brain are involved in regulating ion mobility, the volume fraction of extracellular space and the viscosity of matrix components.

No MeSH data available.