Combining Voltage and Calcium Imaging from Neuronal Dendrites

The ability to monitor membrane potential (V m) and calcium (Ca2+) transients at multiple locations on the same neuron can facilitate further progress in our understanding of neuronal function. Here we describe a method to combine V m and Ca2+ imaging using styryl voltage sensitive dyes and Fura type UV-excitable Ca2+ indicators. In all cases V m optical signals are linear with membrane potential changes, but the calibration of optical signals on an absolute scale is presently possible only in some neurons. The interpretation of Ca2+ optical signals depends on the indicator Ca2+ buffering capacity relative to the cell endogenous buffering capacity. In hippocampal CA1 pyramidal neurons, loaded with JPW-3028 and 300μM Bis-Fura-2, V m optical signals cannot be calibrated and the physiological Ca2+ dynamics are compromised by the presence of the indicator. Nevertheless, at each individual site, relative changes in V m and Ca2+ fluorescence signals under different conditions can provide meaningful new information on local dendritic integration. In cerebellar Purkinje neurons, loaded with JPW-1114 and 1mM Fura-FF, V m optical signals can be calibrated in terms of mV and Ca2+ optical signals quantitatively reveal the physiological changes in free Ca2+. Using these two examples, the method is explained in detai

Functional structure of the mitral cell dendritic tuft in the rat olfactory bulb

Author Posting. © Society for Neuroscience, 2008. This article is posted here by permission of Society for Neuroscience for personal use, not for redistribution. The definitive version was published in Journal of Neuroscience 28 (2008): 4057-4068, doi:10.1523/JNEUROSCI.5296-07.2008.The input–output transform performed by mitral cells, the principal projection neurons of the olfactory bulb, is one of the key factors in understanding olfaction. We used combined calcium and voltage imaging from the same neuron and computer modeling to investigate signal processing in the mitral cells, focusing on the glomerular dendritic tuft. The main finding was that the dendritic tuft functions as a single electrical compartment for subthreshold signals within the range of amplitudes detectable by voltage-sensitive dye recording. These evoked EPSPs had uniform characteristics throughout the glomerular tuft. The Ca2+ transients associated with spatially uniform subthreshold synaptic potentials were comparable but not equal in amplitude in all regions. The average range of normalized amplitudes of the EPSP-driven Ca2+ signals from different locations on dendritic branches in the glomerular tuft was relatively narrow and appeared to be independent of the dendritic surface-to-volume ratio. The computer simulations constrained by the imaging data indicated that a synchronized activation of ~100 synapses randomly distributed on tuft branches was sufficient to generate spatially homogenous EPSPs. This number of activated synapses is consistent with the data from anatomical studies. Furthermore, voltage attenuation of the EPSP along the primary dendrite at physiological temperature was weak compared with other cell types. In the model, weak attenuation of the EPSP along the primary dendrite could be accounted for by passive electrical properties of the mitral cell.This work was supported by National Institutes of Health Grant NS4273

Voltage imaging from dendrites of mitral cells : EPSP attenuation and spike trigger zones

Author Posting. © Society for Neuroscience, 2004. This article is posted here by permission of Society for Neuroscience for personal use, not for redistribution. The definitive version was published in Journal of Neuroscience 24 (2004): 6703-6714, doi:10.1523/JNEUROSCI.0307-04.2004.To obtain a more complete description of individual neurons, it is necessary to complement the electrical patch pipette measurements with technologies that permit a massive parallel recording from many sites on neuronal processes. This can be achieved by using voltage imaging with intracellular dyes. With this approach, we investigated the functional structure of a mitral cell, the principal output neuron in the rat olfactory bulb. The most significant finding concerns the characteristics of EPSPs at the synaptic sites and surprisingly small attenuation along the trunk of the primary dendrite. Also, the experiments were performed to determine the number, location, and stability of spike trigger zones, the excitability of terminal dendritic branches, and the pattern and nature of spike initiation and propagation in the primary and secondary dendrites. The results show that optical data can be used to deduce the amplitude and shape of the EPSPs evoked by olfactory nerve stimulation at the site of origin (glomerular tuft) and to determine its attenuation along the entire length of the primary dendrite. This attenuation corresponds to an unusually large mean apparent "length constant" of the primary dendrite. Furthermore, the images of spike trigger zones showed that an action potential can be initiated in three different compartments of the mitral cell: the soma-axon region, the primary dendrite trunk, and the terminal dendritic tuft, which appears to be fully excitable. Finally, secondary dendrites clearly support the active propagation of action potentialsThis work was supported by National Institutes of Health Grants NS4273 and DC03918. W.R.C. was also supported by the Whitehall Foundation

FHF-independent conduction of action potentials along the leak-resistant cerebellar granule cell axon

Neurons in vertebrate central nervous systems initiate and conduct sodium action potentials in distinct subcellular compartments that differ architecturally and electrically. Here, we report several unanticipated passive and active properties of the cerebellar granule cell’s unmyelinated axon. Whereas spike initiation at the axon initial segment relies on sodium channel (Nav)-associated fibroblast growth factor homologous factor (FHF) proteins to delay Nav inactivation, distal axonal Navs show little FHF association or FHF requirement for high-frequency transmission, velocity and waveforms of conducting action potentials. In addition, leak conductance density along the distal axon is estimated as o1% that of somatodendritic membrane. The faster inactivation rate of FHF-free Navs together with very low axonal leak conductance serves to minimize ionic fluxes and energetic demand during repetitive spike conduction and at rest. The absence of FHFs from Navs at nodes of Ranvier in the central nervous system suggests a similar mechanism of current flux minimization along myelinated axons

Combined voltage and calcium imaging and signal calibration

Voltage imaging using fluorescent voltage-sensitive dyes can be combined with other optical measurements, in particular with Ca2+ imaging, allowing for correlation of membrane potential changes with intracellular Ca2+ signals. Calibration of fluorescence voltage signals permits the comparison of membrane potential changes from different sites allowing spatial mapping of membrane potential changes. These two technical aspects enhance the capability of voltage imaging to address several fundamental problems of neurobiology. Here we discuss how to combine voltage imaging with the optical measurement of intracellular Ca2+ transients and different approaches to calibrate voltage-sensitive dye signals on an absolute scale

Combining Voltage and Calcium Imaging from Neuronal Dendrites

The ability to monitor membrane potential (V (m)) and calcium (Ca(2+)) transients at multiple locations on the same neuron can facilitate further progress in our understanding of neuronal function. Here we describe a method to combine V (m) and Ca(2+) imaging using styryl voltage sensitive dyes and Fura type UV-excitable Ca(2+) indicators. In all cases V (m) optical signals are linear with membrane potential changes, but the calibration of optical signals on an absolute scale is presently possible only in some neurons. The interpretation of Ca(2+) optical signals depends on the indicator Ca(2+) buffering capacity relative to the cell endogenous buffering capacity. In hippocampal CA1 pyramidal neurons, loaded with JPW-3028 and 300 muM Bis-Fura-2, V (m) optical signals cannot be calibrated and the physiological Ca(2+) dynamics are compromised by the presence of the indicator. Nevertheless, at each individual site, relative changes in V (m) and Ca(2+) fluorescence signals under different conditions can provide meaningful new information on local dendritic integration. In cerebellar Purkinje neurons, loaded with JPW-1114 and 1 mM Fura-FF, V (m) optical signals can be calibrated in terms of mV and Ca(2+) optical signals quantitatively reveal the physiological changes in free Ca(2+). Using these two examples, the method is explained in detail

Fast Optical Recordings of Membrane Potential Changes From Dendrites of Pyramidal Neurons

Understanding the biophysical properties of single neurons and how they process information is fundamental to understanding how the brain works. A technique that would allow recording of temporal and spatial dynamics of electrical activity in neuronal processes with adequate resolution would facilitate further research. Here, we report on the application of optical recording of membrane potential transients at many sites on neuronal processes of vertebrate neurons in brain slices using intracellular voltage-sensitive dyes. We obtained evidence that 1) loading the neurons with voltage-sensitive dye using patch electrodes is possible without contamination of the extracellular environment;2) brain slices do not show any autofluorescence at the excitation/emission wavelengths used; 3) pharmacological effects of the dye were completely reversible; 4) the level of photodynamic damage already allows meaningful measurements and could be reduced further; 5) the sensitivity of the dye was comparable to that reported for invertebrate neurons;6) the dye spread ∼500 μm into distal processes within 2 h incubation period. This distance should increase with longer incubation; 7) the optically recorded action potential signals from basolateral dendrites (that are difficult or impossible to approach by patch electrodes) and apical dendrites show that both direct soma stimulation and synaptic stimulation triggered action potentials that originated near the soma. The spikes backpropagated into both basolateral dendrites and apical processes; the propagation was somewhat faster in the apical dendrites

Fast Optical Recordings of Membrane Potential Changes From Dendrites of Pyramidal Neurons

Understanding the biophysical properties of single neurons and how they process information is fundamental to understanding how the brain works. A technique that would allow recording of temporal and spatial dynamics of electrical activity in neuronal processes with adequate resolution would facilitate further research. Here, we report on the application of optical recording of membrane potential transients at many sites on neuronal processes of vertebrate neurons in brain slices using intracellular voltage-sensitive dyes. We obtained evidence that 1) loading the neurons with voltage-sensitive dye using patch electrodes is possible without contamination of the extracellular environment;2) brain slices do not show any autofluorescence at the excitation/emission wavelengths used; 3) pharmacological effects of the dye were completely reversible; 4) the level of photodynamic damage already allows meaningful measurements and could be reduced further; 5) the sensitivity of the dye was comparable to that reported for invertebrate neurons;6) the dye spread ∼500 μm into distal processes within 2 h incubation period. This distance should increase with longer incubation; 7) the optically recorded action potential signals from basolateral dendrites (that are difficult or impossible to approach by patch electrodes) and apical dendrites show that both direct soma stimulation and synaptic stimulation triggered action potentials that originated near the soma. The spikes backpropagated into both basolateral dendrites and apical processes; the propagation was somewhat faster in the apical dendrites

: Ca 2+ and Voltage Imaging

International audienceThe ability to monitor Ca(2+) signals and membrane potential simultaneously at multiple locations on the same neuron facilitates further progress in our understanding of neuronal function. In particular, this method allows correlation of electrical and chemical signals from multiple sites, including those inaccessible to microelectrodes. This protocol describes a procedure for loading cells with two indicators, a Ca(2+)-sensitive Fura dye and voltage-sensitive JPW1114, together with the equipment required for detecting and imaging the two signals. Potential problems are discussed as well as the capabilities and limitations of the technique