Recent Progress in Voltage-Sensitive Dye Imaging for Neuroscience (original) (raw)
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Imaging Spontaneous Neuronal Activity with Voltage‐Sensitive Dyes
Current Protocols, 2021
Accurately mapping changes in cellular membrane potential across large groups of neurons is crucial for understanding the organization and maintenance of neural circuits. Measuring cellular voltage changes by optical means allows greater spatial resolution than traditional electrophysiology methods and is adaptable to high-throughput imaging experiments. VoltageFluors, a class of voltage-sensitive dyes, have recently been used to optically study the spontaneous activity of many neurons simultaneously in dissociated culture. Voltage-Fluors are particularly useful for experiments investigating differences in excitability and connectivity between neurons at different stages of development and in different disease models. The protocols in this article describe general procedures for preparing dissociated cultures, imaging spontaneous activity in dissociated cultures with VoltageFluors, and analyzing optical spontaneous activity data.
Use of voltage-sensitive dyes and optical recordings in the central nervous system
Progress in Neurobiology, 1995
Understanding the spatio-temporal features of the information processing occurring in any complex neural structure requires the monitoring and analysis of the activity in populations of neurons. Electrophysiological and other mapping techniques have provided important insights into the function of neural circuits and neural populations in many systems. However, there remain limitations with these approaches. Therefore, complementary techniques which permit the monitoring of the spatio-temporal activity in neuronal populations are of continued interest. One promising approach to monitor the electrical activity in potmlations of neurons or on multiple sites of a single neuron is with voltage-sensitive dyes coupled with optical r~cording techniques. This review concentrates on the use of voltage-sensitive dyes and optical imaging as tools to study the activity in neuronal populations in the central nervous system. Focusing on 'fast' voltage.sensitive dyes first, several technical issues and developments in optical imaging will be reviewed. These will include more recent developments in voltage-sensitive dyes as well as newer developments in optical recording technology. Second, studies using voltage-sensitive dyes to investigate information processing questions in the central nervous system and in the invertebrate nervous system will be reviewed. Some emphasis will be placed on the cerebellum, but the major goal is to survey how voltage-sensitive dyes and optical recordings have been utilized in the central nervous system. The review will include optical studies on the visual, auditory, olfactory, somatosensory, auditory, hippocampal and brainstem systems, as well as single cell studies addressing information processing questions. Discussion of the intrinsic optical signals is also included. The review attempts to show how voltage-sensitive dyes and optical recordings can be used to obtain high spatial and temporal resolution monitoring of neuronal activity.
In Vivo Voltage-Sensitive Dye Imaging of Subcortical Brain
2015
The whisker system of rodents is an excellent model to study peripherally evoked neural activity in the brain. Discrete neural modules represent each whisker in the somatosensory cortex ("barrels"), thalamus ("barreloids"), and brain stem ("barrelettes"). Stimulation of a single whisker evokes neural activity sequentially in its corresponding barrelette, barreloid, and barrel. Conventional optical imaging of functional activation in the brain is limited to surface structures such as the cerebral cortex. To access subcortical structures and image sensory-evoked neural activity, we designed a needle-based optical system using gradient-index (GRIN) rod lens. We performed voltage-sensitive dye imaging (VSDi) with GRIN rod lens to visualize neural activity evoked in the thalamic barreloids by deflection of whiskers in vivo. We stimulated several whiskers together to determine the sensitivity of our approach in differentiating between different barreloid responses. We also carried out stimulation of different whiskers at different times. Finally, we used muscimol in the barrel cortex to silence the corticothalamic inputs while imaging in the thalamus. Our results show that it is possible to obtain functional maps of the sensory periphery in deep brain structures such as the thalamic barreloids. Our approach can be broadly applicable to functional imaging of other core brain structures.
Voltage-sensitive dyes for monitoring multineuronal activity in the intact central nervous system
1998
Optical monitoring of activity provides new kinds of information about brain function. Two examples are discussed in this article. First, the spike activity of many individual neurons in small ganglia can be determined. Second, the spatiotemporal characteristics of coherent activity in the brain can be directly measured. This article discusses both general characteristics of optical measurements (sources of noise) as well as more methodological aspects related to voltagesensitive dye measurements from the nervous system.
Nature Protocols, 2008
In many brain areas, circuit connectivity is segregated into specific lamina or glomerula. Functional imaging in these anatomically discrete areas is particularly useful in characterizing circuit properties. Voltage-sensitive dye (VSD) imaging directly assays the spatiotemporal dynamics of neuronal activity, including the functional connectivity of the neurons involved. In spatially segregated structures, VSD imaging can define how physiology and connectivity interact, and can identify functional abnormalities in models of neurological and psychiatric disorders. In the following protocol, we describe the in vitro slice preparation, epifluorescence setup and analyses necessary for fast charge-coupled device (CCD)-based VSD imaging combined with simultaneous whole-cell patch recording. The addition of single-cell recordings validates imaging results, and can reveal the relationship between single-cell activity and the VSD-imaged population response; in synchronously activated neurons, this change in whole-cell recorded V m can accurately represent population V m changes driving the VSD responses. Thus, the combined VSD imaging and whole-cell patch approach provides experimental resolution spanning single-cell electrophysiology to complex local circuit responses.
2012
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 voltagesensitive 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 nearoptimal 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.
One of the key approaches for studying neural network function is the simultaneous measurement of the activity of many neurons. Voltage-sensitive dyes (VSDs) simultaneously report the membrane potential of multiple neurons, but often have pharmacological and phototoxic effects on neuronal cells. Yet, to study the homeostatic processes that regulate neural network function long-term recordings of neuronal activities are required. This study aims to test the suitability of the VSDs RH795 and Di-4-ANEPPS for optically recording pattern generating neurons in the stomatogastric nervous system of crustaceans with an emphasis on long-term recordings of the pyloric central pattern generator. We demonstrate that both dyes stain pyloric neurons and determined an optimal concentration and light intensity for optical imaging. Although both dyes provided sufficient signal-to-noise ratio for measuring membrane potentials, Di-4-ANEPPS displayed a higher signal quality indicating an advantage of this dye over RH795 when small neuronal signals need to be recorded. For Di-4-ANEPPS, higher dye concentrations resulted in faster and brighter staining. Signal quality, however, only depended on excitation light strength, but not on dye concentration. RH795 showed weak and slowly developing phototoxic effects on the pyloric motor pattern as well as slow bleaching of the staining and is thus the better choice for long-term experiments. Low concentrations and low excitation intensities can be used as, in contrast to Di-4-ANEPPS, the signal-to-noise ratio was independent of excitation light strength. In summary, RH795 and Di-4-ANEPPS are suitable for optical imaging in the stomatogastric nervous system of crustaceans. They allow simultaneous recording of the membrane potential of multiple neurons with high signal quality. While Di-4-ANEPPS is better suited for short-term experiments that require high signal quality, RH795 is a better candidate for long-term experiments since it has only minor effects on the motor pattern.
Visualization of Cortical Connections with Voltage Sensitive Dyes
1992
We have used voltage-sensitive dyes to monitor in vivo responses to focal electrical stimulation of visual cortex. This technique allows the visualization of local cortical activity patterns as well as foci in distant cortical targets. A novel chamber, headholder, and optical system provided high intensity epifluorescent images of a large cortical expanse while permitting stimulation with a movable microelectrode. Stimulation at moderate intensities (5-100 J.,Lamps) produced afocus ofactivity (typically 1-2 mm width-at-haif-height for rat and monkey) in the vicinity of the stimulating electrode in striate cortex. In addition, there were disjoint foci ofexcitation in extrastriate cortex. The locations of these foci agreed qualitatively with those expected from the known topography of striate and extrastriate cortex. The projection site in monkey V2 had a substructure that was correlated with the known stripe-like modular organization ofV2. We were also able to discern interesting dynamical aspects of the optical activity pattern, including latency differences between the stimulus and projection regions as well as more complex spatio-temporal patterns within each activated region. We believe that this technique, which offers the ability to trace multiple connections in vivo at a resolution comparable to that provided by chemical tracers, is a promising methodfor investigating modular organization and dynamic aspects ofneural connectivity in the cerebral cortex.
Voltage-sensitive dye imaging reveals inhibitory modulation of ongoing cortical activity
2019
Voltage-sensitive dye imaging (VSDI) is a powerful technique for interrogating membrane potential dynamics in assemblies of cortical neurons, but with effective resolution limits that confound interpretation. In particular, it is unclear how VSDI signals relate to population firing rates. To address this limitation, we developed an in silico model of VSDI in a biologically faithful digital reconstruction of rodent neocortical microcircuitry. Using this model, we extend previous experimental observations regarding the cellular origins of VSDI, finding that the signal is driven primarily by neurons in layers 2/3 and 5. We proceed by exploring experimentally inaccessible circuit properties to show that during periods of spontaneous activity, membrane potential fluctuations are anticorrelated with population firing rates. Furthermore, we manipulate network connections to show that this effect depends on recurrent connectivity and is modulated by external input. We conclude that VSDI pri...