Rina Hildesheim

VSDI: a new era in functional imaging of cortical dynamics

During the last few decades, neuroscientists have benefited from the emergence of many powerful functional imaging techniques that cover broad spatial and temporal scales. We can now image single molecules controlling cell differentiation, growth and death; single cells and their neurites processing electrical inputs and sending outputs; neuronal circuits performing neural computations in vitro; and the intact brain. At present, imaging based on voltage-sensitive dyes (VSDI) offers the highest spatial and temporal resolution for imaging neocortical functions in the living brain, and has paved the way for a new era in the functional imaging of cortical dynamics. It has facilitated the exploration of fundamental mechanisms that underlie neocortical development, function and plasticity at the fundmental level of the cortical column.

Imaging Cortical Dynamics at High Spatial and Temporal Resolution with Novel Blue Voltage-Sensitive Dyes

Conventional imaging techniques have provided high-resolution imaging either in the spatial domain or in the temporal domain. Optical imaging utilizing voltage-sensitive dyes has long had the unrealized potential to achieve high resolution in both domains simultaneously, providing subcolumnar spatial detail with millisecond precision. Here, we present a series of developments in voltage-sensitive dyes and instrumentation that make functional imaging of cortical dynamics practical, in both anesthetized and awake behaving preparations, greatly facilitating exploration of the cortex. We illustrate this advance by analyzing the millisecond-by-millisecond emergence of orientation maps in cat visual cortex.

Improved fluorescent probes for the measurement of rapid changes in membrane potential

To improve the quality of fluorescent voltage-sensitive probes twenty new styryl dyes were synthesized. Some of the new probes are significantly better than any used in the past. A signal-to-noise ratio of 90 root mean square (rms) noise was obtained for an optical recording of action potentials from neuroblastoma cells maintained in monolayer culture. The fluorescence fractional change of the optical signal is as large as 14%/100 mV. Photodynamic damage and bleaching are much less significant with the new probes. These fluorescent probes can be used to measure small and rapid changes in membrane potential from single cells maintained in monolayer cultures, from single cells in invertebrate ganglia, from their arborization, and from other preparations. The optical measurement can be made with a standard fluorescent microscope equipped with DC mercury illumination. Guidelines for the design of even better fluorescent probes and more efficient instruments are suggested.

In-vivo Optical Imaging of Cortical Architecture and Dynamics

A number of new imaging techniques are available to scientists to visualize the functioning brain directly, revealing unprecedented details. These imaging techniques have provided a new level of understanding of the principles underlying cortical development, organization and function. In this chapter we will focus on optical imaging in the living mammalian brain, using two complementary imaging techniques. The first technique is based on intrinsic signals. The second technique is based on voltage-sensitive dyes. Currently, these two optical imaging techniques offer the best spatial and temporal resolution, but also have inherent limitations. We shall provide a few examples of new findings obtained mostly in work done in our laboratory. The focus will be upon the understanding of methodological aspects which in turn should contribute to optimal use of these imaging techniques. General reviews describing earlier work done on simpler preparations have been published elsewhere (Cohen, 1973; Tasaki and Warashina, 1976; Waggoner and Grinvald, 1977; Waggoner, 1979; Salzberg, 1983; Grinvald, 1984; Grinvald et al., 1985; De Weer and Salzberg, 1986; Cohen and Lesher, 1986; Salzberg et al., 1986; Loew, 1987; Orbach, 1987; Blasdel, 1988, 1989; Grinvald et al., 1988; Kamino, 1991; Cinelli and Kauer, 1992; Frostig, 1994).

Compartment-resolved imaging of activity-dependent dynamics of cortical blood volume and oximetry.

Optical imaging, positron emission tomography, and functional magnetic resonance imaging (fMRI) all rely on vascular responses to image neuronal activity. Although these imaging techniques are used successfully for functional brain mapping, the detailed spatiotemporal dynamics of hemodynamic events in the various microvascular compartments have remained unknown. Here we used high-resolution optical imaging in area 18 of anesthetized cats to selectively explore sensory-evoked cerebral blood-volume (CBV) changes in the various cortical microvascular compartments. To avoid the confounding effects of hematocrit and oximetry changes, we developed and used a new fluorescent blood plasma tracer and combined these measurements with optical imaging of intrinsic signals at a near-isosbestic wavelength for hemoglobin (565 nm). The vascular response began at the arteriolar level, rapidly spreading toward capillaries and venules. Larger veins lagged behind. Capillaries exhibited clear blood-volume changes. Arterioles and arteries had the largest response, whereas the venous response was smallest. Information about compartment-specific oxygen tension dynamics was obtained in imaging sessions using 605 nm illumination, a wavelength known to reflect primarily oximetric changes, thus being more directly related to electrical activity than CBV changes. Those images were radically different: the response began at the parenchyma level, followed only later by the other microvascular compartments. These results have implications for the modeling of fMRI responses (e.g., the balloon model). Furthermore, functional maps obtained by imaging the capillary CBV response were similar but not identical to those obtained using the early oximetric signal, suggesting the presence of different regulatory mechanisms underlying these two hemodynamic processes.

Fluorescence monitoring of electrical responses from small neurons and their processes.

To improve the sensitivity of fluorescence measurements of electrical responses from small cells and their processes, we have optimized the optical measuring system. The fluorescence intensity from a stained cell was increased 40-fold relative to our previous apparatus. The increased fluorescence intensity permits the use of an inexpensive photodiode (or a photodiode array) that has a approximately 10-fold higher quantum efficiency relative to a photomultiplier. Utilizing the improved apparatus, we optically recorded an action potential of a 2 microns wide neuronal process with a signal-to-noise ratio of approximately 50 (root mean square noise) without averaging. We also report the design of an improved fluorescence voltage-sensitive probe; the fractional change of the fluorescence signal under optimal conditions was 21%/100 mV.

Optical recording of synaptic potentials from processes of single neurons using intracellular potentiometric dyes

To record post synaptic potentials or electrical activity from processes of single cells in a central nervous system (CNS) preparation in situ, voltage sensitive dyes can be injected intracellularly, thereby staining only the cell under investigation. We report the structure, evaluation, and synthesis of 11 fluorescent styryl dyes developed for iontophoretic injection. The optical signals that represent small synaptic potentials from single processes of iontophoretically injected cells are expected to be very small and, therefore, such measurements are not easy. We report the methodology that permitted the optical recording of action potentials from a 3-micron axon and the recording of small synaptic potentials from the processes of single cells in the segmental ganglia of the leech. The same dyes also proved useful for optical recording of action potentials of anterogradely labeled axons, following local extracellular injection at a remote site in a mammalian CNS preparation.

Voltage-Sensitive Dye Imaging of Neocortical Activity

Neural computations underlying sensory perception, cognition, and motor control are performed by populations of neurons at different anatomical and temporal scales. Few techniques are currently available for exploring the dynamics of local and large range populations. Voltage-sensitive dye imaging (VSDI), based on organic voltage probes, reveals neural population activity in areas ranging from a few tens of micrometers to a couple of centimeters, or two areas up to ~10 cm apart. VSDI provides a submillisecond temporal resolution and a spatial resolution of ~50 µm. The dye signal emphasizes subthreshold synaptic potentials. VSDI has been applied in the mouse, rat, gerbil, ferret, tree shrew, cat, and monkey cortices to explore the lateral spread of retinotopic or somatotopic activation; the dynamic spatiotemporal pattern resulting from sensory activation, including the somatosensory, olfactory, auditory, and visual modalities; and motor preparation and the properties of spontaneously occurring population activity. In this introduction, we focus on VSDI in vivo and review results obtained mostly in the visual system in our laboratory.

Dynamic Patterns of Spontaneous Ongoing Activity in the Visual Cortex of Anesthetized and Awake Monkeys are Different

&NA; Ongoing internal cortical activity plays a major role in perception and behavior both in animals and humans. Previously we have shown that spontaneous patterns resembling orientation‐maps appear over large cortical areas in the primary visual‐cortex of anesthetized cats. However, it remains unknown 1) whether spontaneous‐activity in the primate also displays similar patterns and 2) whether a significant difference exists between cortical ongoing‐activity in the anesthetized and awake primate. We explored these questions by combining voltage‐sensitive‐dye imaging with multiunit and local‐field‐potential recordings. Spontaneously emerging orientation and ocular‐dominance maps, spanning up to 6 × 6 mm2, were readily observed in anesthetized but not in awake monkeys. Nevertheless, spontaneous correlated‐activity involving orientation‐domains was observed in awake monkeys. Under both anesthetized and awake conditions, spontaneous correlated‐activity coincided with traveling waves. We found that spontaneous activity resembling orientation‐maps in awake animals spans smaller cortical areas in each instance, but over time it appears across all of V1. Furthermore, in the awake monkey, our results suggest that the synaptic strength had been completely reorganized including connections between dissimilar elements of the functional architecture. These findings lend support to the notion that ongoing‐activity has many more fast switching representations playing an important role in cortical function and behavior.