Voltage-Sensitive Dye Imaging of Population Signals in Brain Slices.

In a bright-field measurement from a vertebrate brain stained by superfusing a solution of the dye over the surface, each pixel in a camera receives light from a substantial number (thousands) of neurons and neuronal processes (population signals). Because of scattering and out-of-focus light, this will be true even if the pixel size corresponds to a small area of the brain. In this situation, the voltage-sensitive dye signal will be a population average of the change in membrane potential of all of these neurons and processes. Many investigators have published voltage-sensitive dye imaging studies of population activities in brain slices. Their methods, including choice of dyes, illumination intensity, and imaging device, vary across a large spectrum. Here we present a protocol for visualizing spatiotemporal patterns in rodent neocortex in vitro. Detecting these patterns requires high-sensitivity imaging in single trials, because averaging will obscure the complex dynamics of the spatiotemporal patterns.

In Vivo Voltage-Sensitive Dye Imaging of Mammalian Cortex Using "Blue" Dyes.

Optical recording of membrane potential allows simultaneous measurements to be taken from many different locations in the nervous system. This is important in studies of the nervous system in which simultaneous activity can occur at the regional, cellular, and subcellular levels. New "blue" dyes, developed by Amiram Grinvald's group, are a great advance for in vivo voltage-sensitive dye imaging of mammalian cortex. The blue dyes are excited by red light (630 nm) that does not overlap with light absorption of hemoglobin (510-590 nm). This virtually eliminates the heart pulsation artifact.

Wide-field and two-photon imaging of brain activity with voltage- and calcium-sensitive dyes.

This chapter presents three examples of imaging brain activity with voltage- or calcium-sensitive dyes. Because experimental measurements are limited by low sensitivity, the chapter then discusses the methodological aspects that are critical for optimal signal-to-noise ratio. Two of the examples use wide-field (1-photon) imaging and the third uses two-photon scanning microscopy. These methods have relatively high temporal resolution ranging from 10 to 10,000 Hz. The three examples are the following: (1) Internally injected voltage-sensitive dye can be used to monitor membrane potential in the dendrites of invertebrate and vertebrate neurons in in vitro preparations. These experiments are directed at understanding how individual neurons convert the complex input synaptic activity into the output spike train. (2) Recently developed methods for staining many individual cells in the mammalian brain with calcium-sensitive dyes together with two-photon microscopy made it possible to follow the spike activity of many neurons simultaneously while in vivo preparations are responding to stimulation. (3) Calcium-sensitive dyes that are internalized into olfactory receptor neurons in the nose will, after several days, be transported to the nerve terminals of these cells in the olfactory bulb glomeruli. There, the population signals can be used as a measure of the input from the nose to the bulb. Three kinds of noise in measuring light intensity are discussed: (1) Shot noise from the random emission of photons from the preparation. (2) Extraneous (technical) noise from external sources. (3) Noise that occurs in the absence of light, the dark noise. In addition, we briefly discuss the light sources, the optics, and the detectors and cameras. The commonly used organic voltage and ion sensitive dyes stain all of the cell types in the preparation indiscriminately. A major effort is underway to find methods for staining individual cell types in the brain selectively. Most of these efforts center around fluorescent protein activity sensors because transgenic methods can be used to express them in individual cell types.