Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Summary

An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.

Abstract

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (V_m-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes ². Many aspects of the initial technology have been continuously improved over several decades ^{3, 5, 11}. Additionally, previous work documented two essential characteristics of V_m-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; ^{10, 14, 16}). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible ^{4, 7}. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects ^{4, 6, 12, 13}. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the V_m-imaging technique. The current sensitivity permits multiple site optical recordings of V_m transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.

Protocol

1. Equipment Setup

Step 1.1. Imaging setup

The key to recording voltage sensitive dye signals is appropriate setup design. We use an upright microscope (BX51WI Olympus or Zeiss AxioExaminer) equipped with three cameras. The setup is designed for illuminating individual neurons in brain slices by excitation light in epi-fluorescence, wide-field microscopy mode using either Nikon 60X/1.0 NA or Zeiss 63X/1.0 NA water dipping objectives. Our microscopes are bolted to a vibration isolation table and equipped with motorized movable stages. Each microscope is equipped with three camera ports. One camera port has a standard h....

Results

Successful confocal microscopy should allow clear identification of intact neuronal processes which are close to the surface of the slice and located in one plane of focus. The selection of nerve cells which are appropriate for voltage imaging prior to voltage-sensitive dye loading is critical. An example of confocal images of L5 pyramidal neurons expressing EGFP in a cortical slice (Crym transgenic mouse line) is shown in Figure 2. Axons of individual neurons are clearly visible. Cells with long intact .......

Discussion

This article describes a voltage-sensitive dye recording method for monitoring electrical activity of individual neurons with sub-micrometer and sub-millisecond spatiotemporal resolution. Laser excitation at near-optimal wavelength (regarding signal size) improved the sensitivity of recording by a factor of ~50 over previous approaches. The current sensitivity enables monitoring electrical signals from all parts of individual neurons, including dendrites, axons, axon collaterals and axon terminals as well as individual d.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Setup components
Upright Microscope	Olympus Inc.	BX51WI	With three camera ports
Motorized Movable Stage	Siskiyou	MXOPi.2
Epi-fluorescence Condenser for Olympus BX51	TILL Photonics	0000-560-11659
Upright Microscope	Carl Zeiss, LLC	AxioExaminer D1	With three camera ports
Motorized Top Plate	Scientifica Limited	MMBP
Epi-fluorescence Condenser for Zeiss Axi–xaminer	TILL Photonics
Data Acquisition Camera	RedShirtImaging LLC	NeuroCCD-SM	High speed, low read noise
CCD for IR-DIC	Dage-MTI	IR-1000
Spinning-Disc Confocal Scanner	Yokogawa	CSU-10
High Spatial Resolution CCD on Confocal Scanner	PCO AG	PixelFly	1392x1024 pixels
DPSS CW Laser (532 Nm)	CNI Optoelectronics Tech. Co., Ltd	MLL-III-532 400mW	Excitation light source
Multi-Mode Fiber Launcher	Siskiyou	SM-CFT
Light Guide	TILL Photonics	0000-515-11524
Shutter	Vincent Associates	LS6
Vibration Isolation Table	Minus k Technology	MK26
Specific reagents
Di-2-ANEPEQ (JPW 1114)	Life Technologies	D-6923	Voltage sensitive dye
Crym-EGFP Mouse Line	GENSAT (MMRRC)	STOCK Tg(Crym-EGFP)GF82Gsat/Mmcd	Sparsely expressing EGFP in Layer 5 cortical neurons