Abstract
The cellular and molecular mechanisms that underlie brain function are challenging to study in the living brain. The development of organotypic slices has provided a welcomed addition to our arsenal of experimental brain preparations by allowing both genetic and prolonged pharmacological manipulations in a system that, much like the acute slice preparation, retains several core features of the cellular and network architecture found in situ. Neurons in organotypic slices can survive in culture for several weeks, can be molecularly manipulated by transfection procedures and their function can be interrogated by traditional cellular electrophysiological or imaging techniques. Here, we describe a cost-effective protocol for the preparation and maintenance of organotypic slices and also describe a protocol for biolistic transfection that can be used to introduce plasmids in a small subset of neurons living in an otherwise molecularly unperturbed network. The implementation of these techniques offers a flexible experimental paradigm that can be used to study a multitude of neuronal mechanisms.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Li CL, McIlwain H (1957) Maintenance of resting membrane potentials in slices of mammalian cerebral cortex and other tissues in vitro. J Physiol 139(2):178–190
Yamamoto C, McIlwain H (1966) Potentials evoked in vitro in preparations from the mammalian brain. Nature 210(5040):1055–1056
Gahwiler BH, Capogna M, Debanne D et al (1997) Organotypic slice cultures: a technique has come of age. Trends Neurosci 20(10): 471–477
Gahwiler BH (1981) Organotypic monolayer cultures of nervous tissue. J Neurosci Methods 4(4):329–342
Stoppini L, Buchs PA, Muller D (1991) A simple method for organotypic cultures of nervous tissue. J Neurosci Methods 37(2):173–182
Noraberg J (2004) Organotypic brain slice cultures: an efficient and reliable method for neurotoxicological screening and mechanistic studies. Altern Lab Anim 32(4):329–337
Schnell E, Sizemore M, Karimzadegan S et al (2002) Direct interactions between PSD-95 and stargazin control synaptic AMPA receptor number. Proc Natl Acad Sci U S A 99(21): 13902–13907
Beique JC, Andrade R (2003) PSD-95 regulates synaptic transmission and plasticity in rat cerebral cortex. J Physiol 546:859–867
Barria A, Malinow R (2005) NMDA receptor subunit composition controls synaptic plasticity by regulating binding to CaMKII. Neuron 48(2):289–301
Beique JC, Imad M, Mladenovic L et al (2007) Mechanism of the 5-hydroxytryptamine 2A receptor-mediated facilitation of synaptic activity in prefrontal cortex. Proc Natl Acad Sci U S A 104(23):9870–9875
Soares C, Lee KF, Nassrallah W et al (2013) Differential subcellular targeting of glutamate receptor subtypes during homeostatic synaptic plasticity. J Neurosci 33(33):13547–13559
Hayashi Y, Shi SH, Esteban JA et al (2000) Driving AMPA receptors into synapses by LTP and CaMKII: requirement for GluR1 and PDZ domain interaction. Science 287(5461): 2262–2267
Buchs PA, Stoppini L, Muller D (1993) Structural modifications associated with synaptic development in area CA1 of rat hippocampal organotypic cultures. Brain Res Dev Brain Res 71(1):81–91
Muller D, Buchs PA, Stoppini L (1993) Time course of synaptic development in hippocampal organotypic cultures. Brain Res Dev Brain Res 71(1):93–100
De Simoni A, Griesinger CB, Edwards FA (2003) Development of rat CA1 neurones in acute versus organotypic slices: role of experience in synaptic morphology and activity. J Physiol 550(Pt 1):135–147
Mellentin C, Møller M, Jahnsen H (2006) Properties of long-term synaptic plasticity and metaplasticity in organotypic slice cultures of rat hippocampus. Exp Brain Res 170(4):522–531
Johnston SA (1990) Biolistic transformation: microbes to mice. Nature 346(6286):776–777
McAllister AK (2000) Biolistic transfection of neurons. Sci STKE 2000(51):pl1
Woods G, Zito K (2008) Preparation of gene gun bullets and biolistic transfection of neurons in slice culture. J Vis Exp (12). pii: 675. doi:10.3791/675
Miller LD, Petrozzino JJ, Mahanty NK et al (1993) Optical imaging of cytosolic calcium, electrophysiology, and ultrastructure in pyramidal neurons of organotypic slice cultures from rat hippocampus. Neuroimage 1(2):109–120
Coyle P (1976) Vascular patterns of the rat hippocampal formation. Exp Neurol 52(3):447–458
O’Brien J, Lummis SC (2002) An improved method of preparing microcarriers for biolistic transfection. Brain Res Brain Res Protoc 10(1): 12–15
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Soares, C., Lee, K.F.H., Cook, D., Béïque, JC. (2014). A Cost-Effective Method for Preparing, Maintaining, and Transfecting Neurons in Organotypic Slices. In: Martina, M., Taverna, S. (eds) Patch-Clamp Methods and Protocols. Methods in Molecular Biology, vol 1183. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1096-0_13
Download citation
DOI: https://doi.org/10.1007/978-1-4939-1096-0_13
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-1095-3
Online ISBN: 978-1-4939-1096-0
eBook Packages: Springer Protocols