Abstract
This protocol details a method for imaging organotypic slice cultures from the mouse hippocampus. The cultures are based on the interface method, which does not require special equipment, is easy to execute, and yields slice cultures that can be imaged repeatedly after they are isolated on postnatal day 6–9 and for up to 6 months in vitro. The preserved tissue architecture facilitates the analysis of defined hippocampal synapses, cells and entire projections. Time-lapse imaging is based on transgenes expressed in the mice, or on constructs introduced through transfection or viral vectors; it can reveal processes that develop over time periods ranging from seconds to months. Imaging can be repeated at least eight times without detectable morphological damage to neurons. Subsequent to imaging, the slices can be processed for immunocytochemistry or electron microscopy, to collect further information about the structures that have been imaged. This protocol can be completed in 35 min.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Conchello, J.A. & Lichtman, J.W. Optical sectioning microscopy. Nat. Methods 2, 920–931 (2005).
Yuste, R. Fluorescence microscopy today. Nat. Methods 2, 902–904 (2005).
Gahwiler, B.H. Organotypic monolayer cultures of nervous tissue. J. Neurosci. Meth. 4, 329–342 (1981).
Stoppini, L., Buchs, P.A. & Muller, D. A simple method for organotypic cultures of nervous tissue. J. Neurosci. Meth. 37, 173–182 (1991).
Gahwiler, B.H. et al. Organotypic slice cultures: a technique has come of age. Trends Neurosci. 20, 471–477 (1997).
De Paola, V., Arber, S. & Caroni, P. AMPA receptors regulate dynamic equilibrium of presynaptic terminals in mature hippocampal networks. Nat. Neurosci. 6, 491–500 (2003).
Galimberti, I. et al. Long-term rearrangements of hippocampal mossy fiber terminal connectivity in the adult regulated by experience. Neuron 50, 749–763 (2006).
Caroni, P. Overexpression of growth-associated proteins in the neurons of adult transgenic mice. J. Neurosci. Meth. 71, 3–9 (1997).
Feng, G. et al. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28, 41–51 (2000).
Araki, R. Transgenic mouse lines expressing synaptopHluorin in hippocampus and cerebellar cortex. Genesis 42, 53–60 (2005).
Lo, D.C., McAllister, A.K. & Katz, L.C. Neuronal transfection in brain slices using particle-mediated gene transfer. Neuron 13, 1263–1268 (1994).
Benediktsson, A.M., Schachtele, S.J., Green, S.H. & Dailey, M.E. Ballistic labeling and dynamic imaging of astrocytes in organotypic hippocampal slice cultures. J. Neurosci. Meth. 141, 41–53 (2005).
Ehrengruber, M.U. et al. Recombinant Semliki Forest virus and Sindbis virus efficiently infect neurons in hippocampal slice cultures. Proc. Natl. Acad. Sci. USA 96, 7041–7046 (1999).
Miyaguchi, K., Maeda, Y., Kojima, T., Setoguchi, Y. & Mori, N. Neuron-targeted gene transfer by adenovirus carrying neural-restrictive silencer element. Neuroreport 10, 2349–2353 (1999).
Henze, D.A., Urban, N.N. & Barrionuevo, G. The multifarious hippocampal mossy fiber pathway: a review. Neuroscience 98, 407–427 (2000).
De Simoni, A., Griesinger, C.B. & Edwards, F.A. Development of rat CA1 neurones in acute versus organotypic slices: role of experience in synaptic morphology and activity. J. Physiol. 550, 135–147 (2003).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Rights and permissions
About this article
Cite this article
Gogolla, N., Galimberti, I., DePaola, V. et al. Long-term live imaging of neuronal circuits in organotypic hippocampal slice cultures. Nat Protoc 1, 1223–1226 (2006). https://doi.org/10.1038/nprot.2006.169
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2006.169
This article is cited by
-
Automated four-dimensional long term imaging enables single cell tracking within organotypic brain slices to study neurodevelopment and degeneration
Communications Biology (2019)
-
Fluidic system for long-term in vitro culturing and monitoring of organotypic brain slices
Biomedical Microdevices (2015)
-
Mouse Delta Opioid Receptors are Located on Presynaptic Afferents to Hippocampal Pyramidal Cells
Cellular and Molecular Neurobiology (2012)
-
Temporally matched subpopulations of selectively interconnected principal neurons in the hippocampus
Nature Neuroscience (2011)
-
In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons
Nature Neuroscience (2010)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.
