Cell and Tissue Research

, Volume 365, Issue 1, pp 29–50 | Cite as

Calcium buffer proteins are specific markers of human retinal neurons

  • Orsolya Kántor
  • Szilvia Mezey
  • Jennifer Adeghate
  • Angela Naumann
  • Roland Nitschke
  • Anna Énzsöly
  • Arnold Szabó
  • Ákos Lukáts
  • János Németh
  • Zoltán Somogyvári
  • Béla Völgyi
Regular Article

Abstract

Ca2+-buffer proteins (CaBPs) modulate the temporal and spatial characteristics of transient intracellular Ca2+-concentration changes in neurons in order to fine-tune the strength and duration of the output signal. CaBPs have been used as neurochemical markers to identify and trace neurons of several brain loci including the mammalian retina. The CaBP content of retinal neurons, however, varies between species and, thus, the results inferred from animal models cannot be utilised directly by clinical ophthalmologists. Moreover, the shortage of well-preserved human samples greatly impedes human retina studies at the cellular and network level. Our purpose has therefore been to examine the distribution of major CaBPs, including calretinin, calbindin-D28, parvalbumin and the recently discovered secretagogin in exceptionally well-preserved human retinal samples. Based on a combination of immunohistochemistry, Neurolucida tracing and Lucifer yellow injections, we have established a database in which the CaBP marker composition can be defined for morphologically identified cell types of the human retina. Hence, we describe the full CaBP make-up for a number of human retinal neurons, including HII horizontal cells, AII amacrine cells, type-1 tyrosine-hydroxylase-expressing amacrine cells and other lesser known neurons. We have also found a number of unidentified cells whose morphology remains to be characterised. We present several examples of the colocalisation of two or three CaBPs with slightly different subcellular distributions in the same cell strongly suggesting a compartment-specific division of labour of Ca2+-buffering by CaBPs. Our work thus provides a neurochemical framework for future ophthalmological studies and renders new information concerning the cellular and subcellular distribution of CaBPs for experimental neuroscience.

Keywords

Calretinin Calbindin Parvalbumin Secretagogin Retina 

Supplementary material

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ESM 1(DOC 73 kb)
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Supplemental Figure 1

Images displaying results of negative control experiments carried out on human retinal cross sections by the omission of primary antibodies and treatment with donkey anti-goat IgG conjugated with DyLight 405 (a, blue), donkey anti-guinea pig conjugated with Cy3 (b, red), donkey anti-rat IgG conjugated with DyLight 649 (c, green), donkey anti-mouse IgG conjugated with Alexa 488 (e, blue), donkey anti-mouse IgG conjugated with DyLight 649 (f, red) and donkey anti-rabbit IgG conjugated with Alexa 555 (g, red) secondary antibodies. Composites of a–c and e–g are shown in d, h. Clearly, the secondary antibodies failed to label any neuronal structure in the human retina and only resulted in some negligible background staining mostly around vessels. Faint autofluorescence of photoreceptor outer segments was also observed. Bars 20 μm. (GIF 50 kb)

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High resolution image (TIF 3378 kb)
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Supplemental Figure 2

Cross sections of the Wistar rat retina stained with antisera utilised in this study. a–d Immunoreactions with goat anti-PV (a, blue), rabbit anti-PV (b, red) and mouse anti-PV (c, green) antisera and a composite image (d). All utilised antibodies resulted in a characteristic staining of PV+ inner retinal cells in the rat retina, including AII amacrine cells. All PV antibodies labelled the same retinal structures. e–h Immunoreactions with mouse anti-CaR (e, blue), goat anti-CaR (f, red) and rabbit anti-CaR (g, green) antisera and a composite image (d). All utilised antibodies resulted in a characteristic staining with CaR+ inner retinal cells in the rat retina, including starburst amacrine cell somata in both the INL and the GCL and three immunolabelled bands in the IPL. i Cross-section of the Wistar rat retina stained with the goat anti-SCGN serum. The antibody specifically stained a population of bipolar cells in the rat retina corresponding strongly to previous descriptions by Puthussery and colleagues (2010). Bars 20 μm (a-h), 10 μm (i). (GIF 194 kb)

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High resolution image (TIF 7868 kb)
441_2016_2376_Fig13_ESM.gif (236 kb)
Supplemental Figure 3

Cross sections of Wistar rat cortex stained with antisera utilised in this study. a–d Low-power photomicrographs of the cortex stained with goat anti-PV (a, blue), rabbit anti-PV (b, red) and mouse anti-PV (c, green) antisera and a composite image (d). Images display PV+ cortical basket cells and their PV+ axon terminals surrounding pyramidal cell somata (asterisks). All sera mark the same neuronal structures. e–h PV+ cortical structures labelled with goat anti-PV (e, blue), rabbit anti-PV (f, red) and mouse anti-PV (g, green) antisera and a composite image (h). i–l Cortical cross-sections labelled with goat anti-CaR (i, blue), rabbit anti-CaR (j, red) and mouse anti-CaR (k, green) antibodies and a composite image (l). Cortical interneurons and neuronal fibres are stained with all three antibodies. Each antiserum labelled the same neuronal elements. Bars 20 μm (a-d), 40 μm (e-l). (GIF 235 kb)

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High resolution image (TIF 7431 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Orsolya Kántor
    • 1
    • 2
  • Szilvia Mezey
    • 1
  • Jennifer Adeghate
    • 1
  • Angela Naumann
    • 3
    • 4
  • Roland Nitschke
    • 3
    • 4
  • Anna Énzsöly
    • 5
    • 6
  • Arnold Szabó
    • 6
  • Ákos Lukáts
    • 6
  • János Németh
    • 5
  • Zoltán Somogyvári
    • 7
  • Béla Völgyi
    • 2
    • 8
    • 9
    • 10
  1. 1.Department of Anatomy, Histology and EmbryologySemmelweis UniversityBudapestHungary
  2. 2.MTA-PTE NAP B Retinal Electrical Synapses Research GroupPécsHungary
  3. 3.Life Imaging Center, Center for Biological Systems AnalysisAlbert Ludwigs UniversityFreiburgGermany
  4. 4.BIOSS Centre for Biological Signaling StudiesAlbert Ludwigs UniversityFreiburgGermany
  5. 5.Department of OphthalmologySemmelweis UniversityBudapestHungary
  6. 6.Department of Human Morphology and Developmental BiologySemmelweis UniversityBudapestHungary
  7. 7.Complex Systems and Computational Neuroscience Group, Wigner Research Center for PhysicsHungarian Academy of SciencesBudapestHungary
  8. 8.Department of Experimental Zoology and NeurobiologyUniversity of PécsPécsHungary
  9. 9.Department of OphthalmologyNew York University Langone Medical CenterNew YorkUSA
  10. 10.Szentágothai Research CenterUniversity of PécsPécsHungary

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