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Calcium buffer proteins are specific markers of human retinal neurons

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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.

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Acknowledgement

The authors thank Zsuzsanna Vidra, Brigitta Fadgyas and Dr. Gábor Baksa for their technical or organisational assistance. The authors are grateful to Dr. Karin Dedek for the sheep secretagogin antibody and to Dr. Mark Eyre for assistance with the English language.

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Authors and Affiliations

  1. Department of Anatomy, Histology and Embryology, Semmelweis University, Budapest, H-1094, Hungary

    Orsolya Kántor, Szilvia Mezey & Jennifer Adeghate

  2. MTA-PTE NAP B Retinal Electrical Synapses Research Group, Pécs, H-7624, Hungary

    Orsolya Kántor & Béla Völgyi

  3. Life Imaging Center, Center for Biological Systems Analysis, Albert Ludwigs University, Freiburg, 79104, Germany

    Angela Naumann & Roland Nitschke

  4. BIOSS Centre for Biological Signaling Studies, Albert Ludwigs University, Freiburg, 79104, Germany

    Angela Naumann & Roland Nitschke

  5. Department of Ophthalmology, Semmelweis University, Budapest, H-1085, Hungary

    Anna Énzsöly & János Németh

  6. Department of Human Morphology and Developmental Biology, Semmelweis University, Budapest, H-1094, Hungary

    Anna Énzsöly, Arnold Szabó & Ákos Lukáts

  7. Complex Systems and Computational Neuroscience Group, Wigner Research Center for Physics, Hungarian Academy of Sciences, Budapest, H-1121, Hungary

    Zoltán Somogyvári

  8. Department of Experimental Zoology and Neurobiology, University of Pécs, Pécs, H-7624, Hungary

    Béla Völgyi

  9. Department of Ophthalmology, New York University Langone Medical Center, New York, NY 10016, USA

    Béla Völgyi

  10. Szentágothai Research Center, University of Pécs, Ifjúság Street 20, H-7624, Pécs, Hungary

    Béla Völgyi

Authors
  1. Orsolya Kántor
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  2. Szilvia Mezey
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  3. Jennifer Adeghate
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  4. Angela Naumann
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  6. Anna Énzsöly
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  7. Arnold Szabó
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  8. Ákos Lukáts
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  9. János Németh
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  10. Zoltán Somogyvári
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  11. Béla Völgyi
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Corresponding author

Correspondence to Béla Völgyi.

Additional information

This work was supported by OTKA K105247 to B.V. and OTKA K-113147 to Z.S. and by the Hungarian Brain Research Program (KTIA_NAP_13-2-2015-0008) to B.V. This research was also supported by the European Union and the State of Hungary, co-financed by the European Social Fund in the framework of the TÁMOP-4.2.4.A/2-11/1-2012-0001 “National Excellence Program” to B.V.

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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)

High resolution image (TIF 3378 kb)

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)

High resolution image (TIF 7868 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)

High resolution image (TIF 7431 kb)

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Kántor, O., Mezey, S., Adeghate, J. et al. Calcium buffer proteins are specific markers of human retinal neurons. Cell Tissue Res 365, 29–50 (2016). https://doi.org/10.1007/s00441-016-2376-z

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