Skip to main content
Springer Nature Link
Log in

Cortical Organotypic Brain Slice Cultures to Examine Sex- and Age-Dependent Astrocyte-Mediated Synaptic Phagocytosis

  • Protocol
  • First Online:
Astrocytes

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2896))

  • 294 Accesses

Abstract

Astrocytes, the most abundant glial cells in the brain, are an integral part of the synaptic compartment and contribute to synaptic pruning, a key process for refining neural circuits during early postnatal development (PND). Dysregulations in this process are implicated in various neuropsychiatric disorders, including major depressive disorder (MDD). To investigate astrocyte functions in a physiologically relevatpdelnt context, organotypic brain slice cultures (OBSCs) offer a powerful model, reproducing more closely in vivo conditions than traditional cell cultures and preserving complex brain architecture and interactions. Here, we present OBSCs as an ex vivo culturing method to provide a platform to explore astrocyte-mediated synaptic pruning dynamics in the rat prefrontal cortex (PFC) during PND. Our approach is based on assessing the role of MEGF10, a key protein involved in synaptic pruning, alongside the synaptic markers synaptophysin and PSD95, using Western blotting to analyze the expression levels of these markers in the cortex of developing rat pups. Additionally, we combine immunofluorescence staining with confocal imaging and IMARIS 9.8 software-assisted analysis to investigate the colocalization of the lysosomal marker LAMP1 with synaptic and astrocytic markers to evaluate the precise rate of synaptic engulfment. The methods presented here allow a deeper examination of an astrocyte-mediated synaptic remodeling in healthy and pathophysiological conditions.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Liu CY, Yang Y, Ju WN, Wang X, Zhang HL (2018) Emerging roles of astrocytes in neuro-vascular unit and the tripartite synapse with emphasis on reactive gliosis in the context of Alzheimer’s disease. Front Cell Neurosci 10(12):193

    Article  Google Scholar 

  2. Sofroniew MV, Vinters HV (2010) Astrocytes: biology and pathology. Acta Neuropathol (Berl) 119(1):7–35

    Article  PubMed  Google Scholar 

  3. Bosworth AP, Allen NJ (2017) The diverse actions of astrocytes during synaptic development. Curr Opin Neurobiol 47:38–43

    Article  CAS  PubMed  Google Scholar 

  4. Allen NJ (2013) Role of glia in developmental synapse formation. Curr Opin Neurobiol 23(6):1027–1033

    Article  CAS  PubMed  Google Scholar 

  5. Stogsdill JA, Eroglu C (2017) The interplay between neurons and glia in synapse development and plasticity. Curr Opin Neurobiol 42:1–8

    Article  CAS  PubMed  Google Scholar 

  6. Eroglu C, Barres BA (2010) Regulation of synaptic connectivity by glia. Nature 468(7321):223–231

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Paumier A, Boisseau S, Jacquier-Sarlin M, Pernet-Gallay K, Buisson A, Albrieux M (2022) Astrocyte–neuron interplay is critical for Alzheimer’s disease pathogenesis and is rescued by TRPA1 channel blockade. Brain 145(1):388–405

    Article  PubMed  Google Scholar 

  8. Bosson A, Boisseau S, Buisson A, Savasta M, Albrieux M (2015) Disruption of dopaminergic transmission remodels tripartite synapse morphology and astrocytic calcium activity within substantia nigra pars reticulata. Glia 63(4):673–683

    Article  PubMed  Google Scholar 

  9. Crunelli V, Carmignoto G, Steinhäuser C (2015) Novel astrocyte targets: new avenues for the therapeutic treatment of epilepsy. Neuroscientist 21(1):62–83

    Article  PubMed  PubMed Central  Google Scholar 

  10. Jia X, Gao Z, Hu H (2021) Microglia in depression: current perspectives. Sci China Life Sci 64(6):911–925

    Article  CAS  PubMed  Google Scholar 

  11. Chung WS, Allen NJ, Eroglu C (2015) Astrocytes control synapse formation, function, and elimination. Cold Spring Harb Perspect Biol 7(9):a020370

    Article  PubMed  PubMed Central  Google Scholar 

  12. Kolb B, Mychasiuk R, Muhammad A, Li Y, Frost DO, Gibb R (2012) Experience and the developing prefrontal cortex. Proc Natl Acad Sci 109(supplement_2):17186–17193

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Drzewiecki CM, Juraska JM (2020) The structural reorganization of the prefrontal cortex during adolescence as a framework for vulnerability to the environment. Pharmacol Biochem Behav 199:173044

    Article  CAS  PubMed  Google Scholar 

  14. Chung WS, Clarke LE, Wang GX, Stafford BK, Sher A, Chakraborty C et al (2013) Astrocytes mediate synapse elimination through MEGF10 and MERTK pathways. Nature 504(7480):394–400

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Bak A, Schmied K, Jakob ML, Bedogni F, Squire OA, Gittel B, Jesinghausen M, Schünemann KD, Weber Y, Kampa B, van Loo KM (2024) Temporal dynamics of neocortical development in organotypic mouse brain cultures: a comprehensive analysis. J Neurophysiol 132(3):1038–1055

    Article  CAS  PubMed  Google Scholar 

  16. Ullrich C, Daschil N, Humpel C (2011) Organotypic vibrosections: novel whole sagittal brain cultures. J Neurosci Methods 201(1):131–141

    Article  PubMed  PubMed Central  Google Scholar 

  17. Vivi E, Seeholzer LR, Nagumanova A, Di Benedetto B (2023) Early age- and sex-dependent regulation of astrocyte-mediated glutamatergic synapse elimination in the rat prefrontal cortex: establishing an organotypic brain slice culture investigating tool. Cells 12(23):2761

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Stoppini L, Buchs PA, Muller D (1991) A simple method for organotypic cultures of nervous tissue. J Neurosci Methods 37(2):173–182

    Article  CAS  PubMed  Google Scholar 

  19. Humpel C (2018) Organotypic brain slice cultures. Curr Protoc Immunol 123(1):e59

    Article  PubMed  Google Scholar 

  20. Thompson D, Odufuwa AE, Watt JA (2023) Microdissection of the supraoptic nucleus and posterior pituitary gland from fresh unfixed tissue. MethodsX 1(11):102388

    Article  Google Scholar 

Download references

Acknowledgments

The authors would like to thank Britta Wenske and Susanne Wallner for their constant support during the establishment of the OBSCs, the execution of the experiments, and critical technical advancements. This work was supported by the Department of Psychiatry and Psychotherapy of the University of Regensburg and by the German Research Council (DFG-GRK2174 “Neurobiology of Emotion Dysfunction” (P1)) to BDB. The FV3000 confocal microscope was funded by a grant (INST 89/506-1 FUGG, 91b GG) from the Deutsche Forschungsgemeinschaft (DFG).

Author information

Authors and Affiliations

  1. Department of Psychiatry and Psychotherapy, University of Regensburg, Regensburg, Germany

    Anastasiia Nagumanova, Lea R. Seeholzer & Barbara Di Benedetto

  2. Regensburg Center of Neuroscience, University of Regensburg, Regensburg, Germany

    Barbara Di Benedetto

Authors
  1. Anastasiia Nagumanova
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lea R. Seeholzer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Barbara Di Benedetto
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Barbara Di Benedetto .

Editor information

Editors and Affiliations

  1. Department of Psychiatry and Psychotherapy, University of Regensburg, Regensburg, Germany

    Barbara Di Benedetto

Rights and permissions

Reprints and permissions

Copyright information

© 2025 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Nagumanova, A., Seeholzer, L.R., Di Benedetto, B. (2025). Cortical Organotypic Brain Slice Cultures to Examine Sex- and Age-Dependent Astrocyte-Mediated Synaptic Phagocytosis. In: Di Benedetto, B. (eds) Astrocytes. Methods in Molecular Biology, vol 2896. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-4366-2_15

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-4366-2_15

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-4365-5

  • Online ISBN: 978-1-0716-4366-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics