Elsevier

Journal of Neuroscience Methods

Volume 218, Issue 2, 15 September 2013, Pages 139-147
Journal of Neuroscience Methods

Basic Neuroscience
Long-lasting single-neuron labeling by in vivo electroporation without microscopic guidance

https://doi.org/10.1016/j.jneumeth.2013.06.004Get rights and content

Highlights

  • We established an experimental protocol for “blind” single-neuron electroporation.

  • Plasmids containing GFP gene were introduced to single neurons by electroporation.

  • GFP expression enabled the structural details of the neuron to be seen clearly.

  • The optimum condition for electroporation was determined electrophysiologically.

  • The labeling by GFP expression persisted at least one month.

Abstract

In order to make a direct link between the morphological and functional study of the nervous system, we established an experimental protocol for labeling individual neurons persistently without microscopic guidance by injecting a plasmid encoding fluorescent protein electroporatively after recording their activity extracellularly. Using a glass pipette filled with electrolyte solution containing a plasmid encoding green fluorescent protein (GFP), single-neuron recording and electroporation were performed on anesthetized rats. When performing the electroporation at the completion of recording, the degree of contact between the target neuron and the electrode tip was adjusted by monitoring the change of the trace of recorded action potentials and the increase of electrode resistance. The expression of GFP and its immunostaining with a polyclonal antibody enabled us to clearly see the basic structural components such as cell bodies, axons, dendrites, and even smaller components such as spines. Identification of the morphological subtypes of neurons was possible with every labeled neuron. The optimum condition for labeling was a 30% increase of the electrode resistance, and the labeling success rate evaluated 3 days after labeling was 40%. The rate evaluated one month after labeling was only slightly lower (33%). We also confirmed experimentally that this recording and labeling procedure can be similarly successful in head-fixed behaving rats. This new experimental protocol will be a breakthrough in systems neuroscience because it makes a direct link between the morphology and behavior-related activity of single neurons.

Introduction

How individual neurons are linked to each other to form circuitries within the nervous system and how their activity leads to complex functions, such as sensory and motor processes as well as learning and cognition, are fundamental questions in neuroscience. With regard to the morphology of individual neurons and neural circuitry, recent technological advancements utilizing virus vectors and fluorescent proteins have enabled us to investigate the divergence of neuron subtypes and the complexity of neural connectivity in the central nervous system much more precisely than ever. With regard to the functions, on the other hand, single-unit recording in behaving animals has long been and still is a major method used to investigate the relation between the activity of individual neurons and behavior. The method was initially established for monkeys (Evarts, 1968) and has recently been being used in studies with other species, such as rodents. There has, however, been no “convenient and easy” way to associate the neuron activity data obtained during behavior with the morphology of those neurons. This is mainly due to the technical difficulties of labeling a neuron from which the activity was recorded. In intracellular recording, a target neuron can be labeled by delivering a label in the inner solution of the electrode to the neuron by iontophoresis, and in patch-clamp recording, the label can be delivered by diffusion. When these techniques are applied to behaving animals, however, maintenance of recording is technically highly demanding and the target neuron is often damaged when an electrode penetrates its membrane (Chorev et al., 2009). Pinault (1996) established the protocol of juxtacellular recording/staining, which is a more efficient technique to monitor the discharge of a neuron and label it afterwards with minimum injury, and this technique has recently been used in studies with anesthetized monkeys (Joshi and Hawken, 2006) and behaving rodents (Isomura et al., 2009, Varga et al., 2012). Furthermore, using in vitro preparation, Graham et al. (2007) established an easier protocol for injecting the dye in much shorter time and combined it with single-unit recording. The major problem faced when using juxtacellular recording/staining in studies with behaving animals is that the dye fades away within only 2 or 3 days. Months-long chronic single-unit recording in trained animals would therefore be impossible if conventional juxtacellular recording/staining were used. An experimenter using the conventional juxtacellular method can obtain data from only a limited number of neurons in an animal that has been trained for a long time, which makes a study quite inefficient or even impossible. This is especially true of studies with monkeys, which for ethical and economic reasons that the number of experimental animals has to be greatly reduced.

The critical factor in the establishment of an efficient method of neuron labeling compatible with chronic single-unit recordings is how long the labeling remains clear. In developmental biology, electroporative delivery of a plasmid encoding fluorescent protein has been proven to be an effective method that can label cells for periods measured in months and has become a standard procedure in the field (Nakamura et al., 2004). Electroporation refers to the cell membrane permeabilization elicited by applying short-duration electric field pulses, traditionally between two relatively large plate electrodes (Ho and Mittal, 1996, Neumann et al., 1999). More recently, single-cell electroporation under microscopic guidance has been developed for in vitro preparations (Lundqvist et al., 1998, Nolkrantz et al., 2001, Rathenberg et al., 2003), and there have been studies describing its application to single cells in vivo (Haas et al., 2001, Kitamura et al., 2008). However, such technique typically needs large-scale setup, such as two-photon microscopy, and even with such a setup the technique cannot be used to label neurons that are more than 1 mm or so below the exposable surface of the brain. In order to label neurons efficiently in various parts of the brain that are more than 1 mm below the exposable surface, the experimental procedure for single-neuron electroporation without microscopic guidance has to be established. Thus we think that “blind” single-neuron electroporation (i.e., without microscopic guidance) could be a technical breakthrough enabling investigators to label individual neurons whose activities were recorded in chronic single-unit recording studies with behaving animals. Furthermore, the staining procedure in single-neuron electroporation is easier and much shorter in time than in the conventional juxtacellular recording/staining and it can deliver any DNA sequence that can be included in a plasmid vector.

Section snippets

Subjects

Forty-eight male albino Wistar rats weighing 230–270 g were used as subjects. They were individually housed under a 12:12, light:dark cycle with light onset at 8:00 P.M. Throughout the experiments, they were treated in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and Tohoku University's guidelines for animal care and use.

Electrophysiological recording and single-neuron electroporation in anesthetized rats

Subjects for in vivo recordings under anesthesia were prepared as follows. Rats were anesthetized by intraperitoneal

Results

In this study, we aimed to establish a standard protocol for in vivo single-neuron electroporation without microscopic guidance, or the “blind” single-neuron electroporation, in head-fixed behaving animals.

After durotomy, the brain was penetrated with a glass pipette electrode filled with standard internal solution containing a plasmid encoding green fluorescent protein (GFP). The electrode penetrated at 10 μm/s and a continuous positive pressure of 50–75 kPa was applied to its cannula until its

Discussion

In this study, we have established an experimental protocol for single-neuron electroporation without microscopic guidance. Electrophysiological recording of the neuron discharge through the glass pipette electrode and the measurement of its tip resistance provided in index of the distance between the target neuron and the electrode tip. When a large positive-going component emerged from a standard positive–negative–positive going spike, the electrode resistance was elevated, which was

Acknowledgments

This study was funded by Grant-in-Aid for Scientific Research (KAKENHI) #24223004, #24243067 and #24120504 to K.T. K.O. was supported by JSPS as a Research Fellow and was funded by KAKENHI #24-8027. We thank Dr. Yoshiko Takahashi (NAIST, Japan) for providing plasmids, pT2K-CAGGS-EGFP. We thank Prof. Takeshi Sakaba for his critical comments on the manuscript.

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These authors contributed equally to this work.

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