Abstract
Here we describe two Neurobiotin electroporation techniques for combined analysis of neuronal structure and function in 300-μm-thick brain slices during development, using mouse hypoglossal (XII) motoneurons as a model. Individual neurons were studied with patch electrodes either in semi-loose (R ∼50–300 MΩ) or in tight-seal (GΩ) configurations. The semi-loose seal allows prolonged recording of spontaneous (if present) or evoked (by voltage steps or extracellular K+) spiking. In tight seal, the cell membrane is first broken down with voltage pulses (50–100 mV). Resulting pore formation in tight-seal mode allows for the stable recording of spontaneous or evoked postsynaptic potentials or currents and the passage of Neurobiotin without spillover into the adjacent cells. With either configuration, Neurobiotin electroporation is achieved by passing 500-ms pulses at 1 Hz (5–25 mV equivalent of ∼300–500 pA) for ∼5 min. Labelling quality of recovered cells is similar between the two configurations; however, the recovery rate of labelled neurons is higher with the semi-loose method. The efficiency and reliability of the semi-loose seal makes it particularly suitable for the serial recording and labelling of multiple neurons within the same preparation. Using semi-loose-seal and tight-seal configurations in combination can increase efficiency and data collected per preparation.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Snow PJ, Rose PK, Brown AG (1976) Tracing axons and axon collaterals of spinal neurons using intracellular injection of horseradish peroxidase. Science 191:312–313
Brown AG, Rose PK, Snow PJ (1977) The morphology of spinocervical tract neurones revealed by intracellular injection of horseradish peroxidase. J Physiol 270:747–764
Brown AG, Fyffe RE (1984) Intracellular staining of mammalian neurons. Academic, London
Horikawa K, Armstrong WE (1988) A versatile means of intracellular labeling: injection of biocytin and its detection with avidin conjugates. J Neurosci Methods 25:1–11
Imanaga I, Kameyama M, Irisawa H (1987) Cell-to-cell diffusion of fluorescent dyes in paired ventricular cells. Am J Physiol 252:H223–H232
Werblin F, Maguire G, Lukasiewicz P, Eliasof S, Wu SM (1988) Neural interactions mediating the detection of motion in the retina of the tiger salamander. Vis Neurosci 1:317–329
Edwards FA, Konnerth A, Sakmann B, Takahashi T (1989) A thin slice preparation for patch clamp recordings from neurones of the mammalian central nervous system. Pflugers Arch 414:600–612
Pinault D (1996) A novel single-cell staining procedure performed in vivo under electrophysiological control: morpho-functional features of juxtacellularly labeled thalamic cells and other central neurons with biocytin or Neurobiotin. J Neurosci Methods 65:113–136
Brock LG, Coombs JS, Eccles JC (1952) The recording of potentials from motoneurons with an intracellular electrode. J Physiol 117:431–460
Stewart WW (1978) Functional connections between cells as revealed by dye-coupling with a highly fluorescent naphthalimide tracer. Cell 14:741–759
Kita H, Armstrong W (1991) A biotin-containing compound N-(2-aminoethyl)biotinamide for intracellular labeling and neuronal tracing studies: comparison with biocytin. J Neurosci Methods 37:141–150
Vaney DI (1991) Many diverse types of retinal neurons show tracer coupling when injected with biocytin or Neurobiotin. Neurosci Lett 125:187–190
Lipski J, Zhang X, Kruszewska B, Kanjhan R (1994) Morphological study of long axonal projections of ventral medullary inspiratory neurons in the rat. Brain Res 640:171–184
Aghajanian GK, Rasmussen K (1989) Intracellular studies in the facial nucleus illustrating a simple new method for obtaining viable motoneurons in adult rat brain slices. Synapse 3:331–338
Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 391:85–100
Schreihofer AM, Guyenet PG (1997) Identification of C1 presympathetic neurons in rat rostral ventrolateral medulla by juxtacellular labeling in vivo. J Comp Neurol 387:524–536
Pilowsky PM, Makeham J (2001) Juxtacellular labeling of identified neurons: kiss the cells and make them dye. J Comp Neurol 433:1–3
Kanjhan R, Vaney DI (2008) Semi-loose seal Neurobiotin electroporation for combined structural and functional analysis of neurons. Pflugers Arch 457:561–568
Bellingham MC, Berger AJ (1996) Presynaptic depression of excitatory synaptic inputs to rat hypoglossal motoneurons by muscarinic M2 receptors. J Neurophysiol 76:3758–3770
Neher E, Sakmann B, Steinbach JH (1978) The extracellular patch clamp: a method for resolving currents through individual open channels in biological membranes. Pflugers Arch 375:219–228
Roberts WM, Almers W (1992) Patch voltage clamping with low-resistance seals: loose patch clamp. Methods Enzymol 207:155–176
Mazza E, Nunez-Abades PA, Spielmann JM, Cameron WE (1992) Anatomical and electrotonic coupling in developing genioglossal motoneurons of the rat. Brain Res 598:127–137
Acknowledgements
We are grateful to David Vaney for his generous support. Matthew Fogarty and Luke Hammond are thanked for their help with confocal imaging. The project was supported by grants to MB from the Australian National Health and Medical Research Council (569827), the Australian Brain Foundation, and the Motor Neurone Disease Research Institute of Australia.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Kanjhan, R., Bellingham, M.C. (2013). Neurobiotin Electroporation for Combined Structural and Functional Analysis of Neurons in Developing Mouse Brain Slices. In: Pilowsky, P., Farnham, M., Fong, A. (eds) Stimulation and Inhibition of Neurons. Neuromethods, vol 78. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-233-9_9
Download citation
DOI: https://doi.org/10.1007/978-1-62703-233-9_9
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-232-2
Online ISBN: 978-1-62703-233-9
eBook Packages: Springer Protocols