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Intracellular and Juxtacellular Staining with Biocytin

Charles J. Wilson¹, R.N.S. Sachdev¹

¹The University of Texas at San Antonio, San Antonio, Texas

Publication Name:

Current Protocols in Neuroscience

Unit Number:

Unit 1.12

DOI:

10.1002/0471142301.ns0112s26

Online Posting Date:

May, 2004

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Abstract

Many physiological studies require microscopic examination of the recorded neuron for identification. This unit describes how intracellular and extracellular recording can be combined with single-neuron staining to enable sequential physiological and morphological studies.

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Unit Introduction
Basic Protocol 1: Intracellular Biocytin Staining with Sharp Electrodes
Alternate Protocol: Intracellular Biocytin Staining with Patch Electrodes
Basic Protocol 2: Juxtacellular Biocytin Staining
Support Protocol: Tissue Processing
Commentary
Literature Cited
Figures

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Materials

Support Protocol: Tissue Processing

Materials

Tissues
0.1% Triton-X 100
Phosphate-buffered saline (PBS), pH 7.2
ABC kit or avidin-conjugated fluorescent marker (e.g., avidin-Texas Red)
0.05% (v/v) 3¢,3¢-diaminobenzidine (DAB)
3% (v/v) hydrogen peroxide

Vibratome or freezing microtome
Shaker
Additional reagents and equipment for dehydrating and mounting sections (unit 1.1)

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Figures

Figure 1.12.1

Biocytin-filled cell. This is a pyramidal cell recorded in layer V of primary somatosensory cortex with electrodes filled with 3% biocytin and 1 M potassium acetate. The cell was stable for 1.5 hr, and was stained for 40 min with 1-nA current pulses (100 msec) delivered at 3 Hz. The axon of this neuron was followed out of cortex through the striatum to a terminal site in the perirhinal cortex. Scale bar = 50 µm.

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Literature Cited

Literature Cited
	Bevan, M.D., Booth, P.A., Eaton, S.A., and Bolam, J.P. 1998. Selective innervation of neostriatal interneurons by a subclass of neuron in the globus pallidus of the rat. J. Neurosci. 18:9438-9452.
	Horikawa, K. and Armstrong W. 1988. A versatile means of intracellular labeling: Injection of biocytin and its detection with avidin conjugates. J. Neurosci. Methods 25:1-11.
	Kawaguchi, Y., Wilson, C.J., and Emson, P.C. 1989. Intracellular recording of identified neostriatal patch and matrix spiny cells in a slice preparation preserving cortical inputs. J. Neurophysiol. 62:1052-1068.
	Kawaguchi, Y. 1992. Large aspiny cells in the matrix of the rat neostriatum in vitro: Physiological identification, relation to the compartments and excitatory postsynaptic currents. J. Neurophysiol. 67:1669-1682.
	Kawaguchi, Y. 1993. Physiological, morphological, and histochemical characterization of three classes of interneurons in rat neostriatum. J. Neurosci. 13:4908-4923.
	Nisenbaum, E.S. and Wilson, C.J. 1995. Potassium currents responsible for inward and outward rectification in rat neostriatal spiny projection neurons. J. Neurosci. 15:4449-4463.
	Pinault, D. 1994. Golgi-like labeling of a single neuron recorded extracellularly. Neurosci. Lett. 11:255-260
	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.
	Xu, Z.C., Wilson, C.J., and Emson, P.C. 1992. Morphology of intracellularly stained spiny neurons in rat striatal grafts. Neuroscience 48:95-110.
	Zheng, T. and Wilson, C.J. 2002. Corticostriatal combinatorics: The implications of corticostriatal axonal arborizations. J. Neurophysiol. 87:1007-1017.