Name: juxtasomal biocytin labeling to study the structure-function relationship of individual cortical neurons
Uploaded: 2014-02-25T14:45:00
Description: Watch this Scientific Journal Video about Juxtasomal Biocytin Labeling to Study the Structure-function Relationship of Individual Cortical Neurons at JoVE.com

We would like to thank Profs. Huibert Mansvelder and Bert Sakmann for extensive support, Dr. Marcel Oberlaender for fruitful discussions and providing neuronal tracing, and Brendan Lodder for technical assistance. Data was acquired using the ntrode VI for LabView, generously provided by R. Bruno (Columbia Univ., NY, USA). This research was supported by the Max Planck Society and the Bernstein Center for Computational Neuroscience, Tuebingen (funded by the German Federal Ministry of Education and Research (BMBF; FKZ: 01GQ1002))( R.T.N.), Center for Neurogenomics and Cognitive Research (CNCR), Neuroscience Campus Amsterdam (NCA), funding to C.P.J.d.K. (NWO-ALW #822.02.013 and ENC-Network #p3-c3) and the VU University Amsterdam.

1. Preparation of the Animal
All experimental procedures are carried out in accordance with the Dutch law and after evaluation by a local ethical committee at the VU University Amsterdam, The Netherlands.
<ol>
	<li>Anesthetize a Wistar rat (P25-P45, &#9794;/&#9792;) with isoflurane (2-3% in oxygen) and subsequently with urethane (20% in 0.9% NaCl, 1.6-1.7 g/kg) by intraperitoneal injection. Assess the depth of the anesthesia by monitoring pinch withdrawal, eyelid reflexes, and vibrissae move...

<ol>
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The juxtasomal method allows recording in vivo action potential spiking from single units across behavioral conditions (anesthetized, awake head-fixed or freely moving) with the option of biocytin-labeling the recorded neuron for post&#160;hoc cell type classification and/or 3D reconstruction. The major advantage is to obtain physiological parameters in the extracellular (thus noninvasive) configuration, yet being able to label the neuron intracellularly with biocytin16,17,32. In addition to ...

Neuronal networks consist of multiple cell types, characterized by highly specific morphological and physiological properties1-7. As a consequence, individual cell types perform specialized tasks within the network (see for instance&#160;Gentet&#160;et al.8 and&#160;Burgalossi&#160;et al.9). 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 obtained1,10-15. Here, we demonstrate the juxtasomal labeling technique16,17 which involves electrophysiological recordings using conventional patch pipettes in the extracellular (thus noninvasive) configuration in combination with electroporation of the recorded neuron with biocytin. The major advantage of this approach is that the noninvasive nature ensures that action potential spiking of individual neurons is recorded without altering (e.g. dialyzing) the intracellular content of the cell. Followed by electroporation, the juxtasomal approach provides the option of post&#160;hoc cell identification and reconstruction to link function (physiology) to structure (morphology). Typically, morphological reconstruction involves reconstruction of dendritic and axonal morphology which can be extended to quantification of spine and/or bouton densities or even reconstruction of neuronal morphology at nanometer resolution using electron microscopy. The juxtasomal recording technique can be used for in vivo recordings of various cell-types across cortical layers or in sub-cortical areas in a range of species, although most studies have applied the technique in small rodents such as mice or rats. Our research is focused on recording and labeling neurons from rat primary somatosensory cortex (S1) and involves visual identification of recorded neurons18, dendritic reconstructions in combination with precise registration in a standardized reference frame to reverse engineer cortical networks4,19 and detailed reconstruction of axonal architecture to characterize cell type-specific local and long-range projection targets20.
Compared to alternative in vivo recording techniques (intracellular or whole-cell), juxtasomal recordings are relatively stable and can therefore be applied across behavioral states including anesthetized21,22, sedated14, awake head-fixed23, or even freely-moving animals9. Here, we show juxtasomal labeling in S1 of a urethane-anesthetized rat, although we emphasize the general applicability of this technique to many preparations of choice.

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		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Trizma base</td>
			<td style="width:275px;">Sigma</td>
			<td style="width:119px;">T4661</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Mowiol 4-88</td>
			<td style="width:275px;">Aldrich</td>
			<td style="width:119px;">81381</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Analytical grade glycerol</td>
			<td style="width:275px;">Fluka</td>
			<td style="width:119px;">49767</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">HEPES</td>
			<td style="width:275px;">Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">NaCl</td>
			<td style="width:275px;">Sigma Aldrich</td>
			<td style="width:119px;">31434</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">KCl</td>
			<td style="width:275px;">Sigma Aldrich</td>
			<td style="width:119px;">60130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">CaCl</td>
			<td style="width:275px;">Sigma Aldrich</td>
			<td style="width:119px;">22,350-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">MgCl2</td>
			<td style="width:275px;">Fluka</td>
			<td style="width:119px;">63072</td>
			<td></td>
		</tr>
	</tbody>
</table>

juxtasomal biocytin labeling to study the structure-function relationship of individual cortical neurons

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&#160;hoc identifying the recorded neurons. Here, we demonstrate the juxtasomal biocytin labeling technique, which involves recording action potential spiking in the extracellular (or loose-patch) 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&#160;hoc identification and morphological reconstruction.

Detailed knowledge on 3D structure of individual neurons is crucial for elucidating organizational principles of neuronal networks. Our method involves a pipeline to achieve high quality biocytin labeling from an in vivo preparation, thereby allowing post&#160;hoc neuronal classification and detailed reconstruction of dendritic and axonal architecture of single neurons at high resolution. Depending on the quality of juxtasomal labeling, neurons are recovered with different DAB-intensities ranging from f...

Watch this Scientific Journal Video about Juxtasomal Biocytin Labeling to Study the Structure-function Relationship of Individual Cortical Neurons at JoVE.com

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

To understand the structure of neuronal networks, functional and morphological characterization of individual neurons is a necessity. Here, we demonstrate juxtasomal biocytin labeling, which allows electrophysiological recordings in the extracellular configuration, yet maintaining the ability to intracellularly label the neuron for post&#160;hoc reconstruction of dendritic and axonal architecture.

The cerebral cortex is characterized by multiple layers and many distinct cell-types that together as a network are responsible for many higher cognitive ...

Research

JoVE Journal

Neuroscience

Laser Capture Microdissection of Drosophila Peripheral Neurons

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using the ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

Optogenetic methods  have emerged as a powerful tool for elucidating neural circuit activity  underlying a diverse set of behaviors across a broad range ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...