Improved biocytin labeling and neuronal 3D reconstruction

Marx, Manuel; Günter, Robert Heinz; Hucko, Werner; Radnikow, Gabriele; Feldmeyer, Dirk

doi:10.1038/nprot.2011.449

Cited by 86 publications

(84 citation statements)

References 72 publications

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“…Prior to embedding, transfer slides in solutions with increasing concentrations of ethanol (20 -100%) in order to dehydrate them. NOTE: The dehydration process should be slow otherwise distortions in the dendritic and axonal processes are likely to occur 37 . If an alternative embedding procedure is chosen, e.g., one with the glycerol-based Moviol, a dehydration is not required; however this is likely to result in an inhomogeneous compression of the slice and hence a distorted neuronal morphology.…”

Section: Paired Patch-clamp Recording and Biocytin Fillingmentioning

confidence: 99%

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Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

Qi¹,

Radnikow²,

Feldmeyer

2015

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The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with simultaneous intracellular biocytin filling allows a correlated analysis of their structural and functional properties. With this method it is possible to identify and characterize both pre- and postsynaptic neurons by their morphology and electrophysiological response pattern. Paired recordings allow studying the connectivity patterns between these neurons as well as the properties of both chemical and electrical synaptic transmission. Here, we give a step-by-step description of the procedures required to obtain reliable paired recordings together with an optimal recovery of the neuron morphology. We will describe how pairs of neurons connected via chemical synapses or gap junctions are identified in brain slice preparations. We will outline how neurons are reconstructed to obtain their 3D morphology of the dendritic and axonal domain and how synaptic contacts are identified and localized. We will also discuss the caveats and limitations of the paired recording technique, in particular those associated with dendritic and axonal truncations during the preparation of brain slices because these strongly affect connectivity estimates. However, because of the versatility of the paired recording approach it will remain a valuable tool in characterizing different aspects of synaptic transmission at identified neuronal microcircuits in the brain.

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Section: Paired Patch-clamp Recording and Biocytin Fillingmentioning

confidence: 99%

“…Shrinkage correction factors for the embedding medium are ~1.1 for x and y directions and ~2.1 for the z-direction 37 . 6.…”

Section: Neuronal Reconstruction and Synaptic Contact Localizationmentioning

confidence: 99%

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

Qi¹,

Radnikow²,

Feldmeyer

2015

JoVE

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“…Useful tricks on histochemical procedures are in Refs. 58,59 7. Post Hoc Visualization of Biocytin-filled Neurons 1.…”

Section: Biocytin Labeling Of Nigral Neuronsmentioning

confidence: 99%

“…Protect the slices from light throughout the staining. As an alternative to fluorescence, label neurons via 3,3'-diaminobenzidine for light or electron microscopic analysis 21,58 . CAUTION: Triton X-100 is dangerous.…”

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confidence: 99%

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Engel

2016

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Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

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“…We are only beginning to understand cell type-specific functions across neuronal networks and much is still to be discovered. To this end, many labs are implementing experimental approaches that allow the analysis of morphological properties of the same neuronal population from which physiological parameters have been obtained 1,[10][11][12][13][14][15] . Here, we demonstrate the juxtasomal labeling technique 16,17 which involves electrophysiological recordings using conventional patch pipettes in the extracellular (thus noninvasive) configuration in combination with electroporation of the recorded neuron with biocytin.…”

Section: Introductionmentioning

confidence: 99%

Juxtasomal Biocytin Labeling to Study the Structure-function Relationship of Individual Cortical Neurons

Narayanan

Mohan

Broersen

et al. 2014

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The cerebral cortex is characterized by multiple layers and many distinct cell-types that together as a network are responsible for many higher cognitive functions including decision making, sensory-guided behavior or memory. To understand how such intricate neuronal networks perform such tasks, a crucial step is to determine the function (or electrical activity) of individual cell types within the network, preferentially when the animal is performing a relevant cognitive task. Additionally, it is equally important to determine the anatomical structure of the network and the morphological architecture of the individual neurons to allow reverse engineering the cortical network. Technical breakthroughs available today allow recording cellular activity in awake, behaving animals with the valuable option of post hoc identifying the recorded neurons. Here, we demonstrate the juxtasomal biocytin labeling technique, which involves recording action potential spiking in the extracellular (or loosepatch) configuration using conventional patch pipettes. The juxtasomal recording configuration is relatively stable and applicable across behavioral conditions, including anesthetized, sedated, awake head-fixed, and even in the freely moving animal. Thus, this method allows linking cell-type specific action potential spiking during animal behavior to reconstruction of the individual neurons and ultimately, the entire cortical microcircuit. In this video manuscript, we show how individual neurons in the juxtasomal configuration can be labeled with biocytin in the urethane-anaesthetized rat for post hoc identification and morphological reconstruction. Video LinkThe video component of this article can be found at

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Improved biocytin labeling and neuronal 3D reconstruction

Cited by 86 publications

References 72 publications

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Juxtasomal Biocytin Labeling to Study the Structure-function Relationship of Individual Cortical Neurons

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