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Super-resolution Microscopical Localization of Dopamine Receptors 1 and 2 in Rat Hippocampal Synaptosomes

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Abstract

Although dopamine receptors D1 and D2 play key roles in hippocampal function, their synaptic localization within the hippocampus has not been fully elucidated. In order to understand precise functions of pre- or postsynaptic dopamine receptors (DRs), the development of protocols to differentiate pre- and postsynaptic DRs is essential. So far, most studies on determination and quantification of DRs did not discriminate between subsynaptic localization. Therefore, the aim of the study was to generate a robust workflow for the localization of DRs. This work provides the basis for future work on hippocampal DRs, in light that DRs may have different functions at pre- or postsynaptic sites. Synaptosomes from rat hippocampi isolated by a sucrose gradient protocol were prepared for super-resolution direct stochastic optical reconstruction microscopy (dSTORM) using Bassoon as a presynaptic zone and Homer1 as postsynaptic density marker. Direct labeling of primary validated antibodies against dopamine receptors D1 (D1R) and D2 (D2R) with Alexa Fluor 594 enabled unequivocal assignment of D1R and D2R to both, pre- and postsynaptic sites. D1R immunoreactivity clusters were observed within the presynaptic active zone as well as at perisynaptic sites at the edge of the presynaptic active zone. The results may be useful for the interpretation of previous studies and the design of future work on DRs in the hippocampus. Moreover, the reduction of the complexity of brain tissue by the use of synaptosomal preparations and dSTORM technology may represent a useful tool for synaptic localization of brain proteins.

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Acknowledgements

We are highly indebted to Prof. Dr. F. Monje-Quiroga, Medical University of Vienna, for providing know-how and facility for neuronal culture studies.

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Author notes

  1. Andras G. Miklosi and Giorgia Del Favero contributed equally to this publication.

Authors and Affiliations

  1. Department of Pharmaceutical Chemistry, University of Vienna, 1090, Vienna, Austria

    Andras G. Miklosi, Tanja Bulat & Gert Lubec

  2. Molecular Biotechnology Section, FH Campus Wien, University of Applied Sciences, 1030, Vienna, Austria

    Andras G. Miklosi

  3. Department of Food Chemistry and Toxicology, University of Vienna, 1090, Vienna, Austria

    Giorgia Del Favero & Doris Marko

  4. Core Unit of Biomedical Research, Division of Laboratory Animal Science and Genetics, Medical University of Vienna, 2523, Himberg, Austria

    Harald Höger

  5. Institute of Science and Technology Austria, 3400, Klosterneuburg, Austria

    Ryuichi Shigemoto

Authors
  1. Andras G. Miklosi
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  2. Giorgia Del Favero
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  3. Tanja Bulat
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  7. Gert Lubec
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Corresponding author

Correspondence to Gert Lubec.

Electronic supplementary material

Supplemental Figure 1

Figures showing the profile of Bassoon (red) and Homer1 (green) signal in synaptosomes (a) and primary neuron cultures (b) measured along the trans-synaptic axis. The values used to determine distances between synaptic markers were obtained through these measurements. (GIF 204 kb)

High-resolution image (TIFF 3860 kb)

Supplemental Table 1

Identification of all proteins found in the synaptosomal preparation by mass spectrometric analysis (XLSX 130 kb)

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Miklosi, A.G., Del Favero, G., Bulat, T. et al. Super-resolution Microscopical Localization of Dopamine Receptors 1 and 2 in Rat Hippocampal Synaptosomes. Mol Neurobiol 55, 4857–4869 (2018). https://doi.org/10.1007/s12035-017-0688-y

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  • DOI: https://doi.org/10.1007/s12035-017-0688-y

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