Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Summary

Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.

Abstract

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)¹. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue². Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (>97% spike detection efficiency), has a low rate of false positive spike detection (< 0.003 spikes/sec), and a high temporal precision (about 3 msec) ³. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo^3,4.

Protocol

1. Optical Setup (Figure 1)

1. For two-photon excitation an infrared pulsed laser system with femtosecond pulses is used. A high laser output power (in some cases >2W at 890 nm wavelength) is required to offset the large losses introduced by the optical components of the system.
2. A prechirper system consisting of two prisms imparts a negative group velocity dispersion (GVD) onto the laser pulses prior to the acousto-optical deflectors (AODs) to compensate for the temporal dispersion introduced by the AODs¹.
3. Two AODs with large apertures (10 mm for a 40x water immersion objective with NA 0.8) deflect the laser beam in two dimensio....

Discussion

Dithered random-access scanning indirectly detects suprathreshold spiking activity from the increases in intracellular somatic calcium associated with each spike in a neuron somata. The increases in intracellular calcium are detected by fluorescent calcium dyes. The limitations of dithered random-access scanning arise largely from of the limited signal-to-noise ratio of the calcium fluorescence signals. The signal-to-noise ratio is in turn limited by photodamage, which does not allow using high excitation rates. Because .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Optical components are listed in order, starting from the laser
Titan:Sapphire Laser	Coherent Inc.	Chameleon Ultra 2	High power output recommended (>2W at 900 nm)
Achromatic lens f = 30 mm	Thor labs	AC254-030-B	Anti-reflection (AR) coating for 650-1050 nm
Achromatic lens f = 100 mm	Thor labs	AC254-100-B	AR 650-1050 nm
lens f = 75 mm	Thor labs	LA1608-B	AR 650-1050 nm
lens f = 175 mm	Thor labs	LA1229-B	AR 650-1050 nm
Achromatic lens f = 300 mm	Thor labs	AC254-300-B	AR 650-1050 nm
Achromatic lens f = 100 mm	Thor labs	AC254-100-B	AR 650-1050 nm
Achromatic lens f = 100 mm	Thor labs	AC254-100-B	AR 650-1050 nm
Acousto-optical deflectors	Intraaction Corp	ATD 6510CD2
Reflective diffraction grating	Newport	53-011R	100 grooves/mm for AODs with 65 MHz bandwidth and scan angle of 45 mrad
21.6 mm Brewster prisms	Lambda Research Optics Inc.	IBP21.6SF10
Colored Glass	Schott	BG-39
Dichroic mirror	Chroma Technology Corp	Z532RDC
Photomultiplier modules	Hamamatsu	H9305-03
DAC-ADC board	National Instruments	PCI-6115
Oregon Green 488 Bapta-1 AM	Invitrogen	O-6807