520 research outputs found
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
Normalization of Voltage-Sensitive Dye Signal with Functional Activity Measures
In general, signal amplitude in optical imaging is normalized using the
well-established ΔF/F method, where functional activity is divided by
the total fluorescent light flux. This measure is used both directly, as a
measure of population activity, and indirectly, to quantify spatial and
spatiotemporal activity patterns. Despite its ubiquitous use, the stability and
accuracy of this measure has not been validated for voltage-sensitive dye
imaging of mammalian neocortex in vivo. In this report, we find
that this normalization can introduce dynamic biases. In particular, the
ΔF/F is influenced by dye staining quality, and the ratio is also
unstable over the course of experiments. As methods to record and analyze
optical imaging signals become more precise, such biases can have an
increasingly pernicious impact on the accuracy of findings, especially in the
comparison of cytoarchitechtonic areas, in area-of-activation measurements, and
in plasticity or developmental experiments. These dynamic biases of the
ΔF/F method may, to an extent, be mitigated by a novel method of
normalization, ΔF/ΔFepileptiform. This normalization
uses as a reference the measured activity of epileptiform spikes elicited by
global disinhibition with bicuculline methiodide. Since this normalization is
based on a functional measure, i.e. the signal amplitude of
“hypersynchronized” bursts of activity in the cortical
network, it is less influenced by staining of non-functional elements. We
demonstrate that such a functional measure can better represent the amplitude of
population mass action, and discuss alternative functional normalizations based
on the amplitude of synchronized spontaneous sleep-like activity. These findings
demonstrate that the traditional ΔF/F normalization of voltage-sensitive
dye signals can introduce pernicious inaccuracies in the quantification of
neural population activity. They further suggest that normalization-independent
metrics such as waveform propagation patterns, oscillations in single detectors,
and phase relationships between detector pairs may better capture the biological
information which is obtained by high-sensitivity imaging
ANNINE-6plus, a voltage-sensitive dye with good solubility, strong membrane binding and high sensitivity
We present a novel voltage-sensitive hemicyanine dye ANNINE-6plus and describe its synthesis, its properties and its voltage-sensitivity in neurons. The dye ANNINE-6plus is a salt with a double positively charged chromophore and two bromide counterions. It is derived from the zwitterionic dye ANNINE-6. While ANNINE-6 is insoluble in water, ANNINE-6plus exhibits a high solubility of around 1 mM. Nonetheless, it displays a strong binding to lipid membranes. In contrast to ANNINE-6, the novel dye can be used to stain cells from aqueous solution without surfactants or organic solvents. In neuronal membranes, ANNINE-6plus exhibits the same molecular Stark effect as ANNINE-6. As a consequence, a high voltage-sensitivity is achieved with illumination and detection in the red end of the excitation and emission spectra, respectively. ANNINE-6plus will be particularly useful for sensitive optical recording of neuronal excitation when organic solvents and surfactants must be avoided as with intracellular or extracellular staining of brain tissue
Understanding visual map formation through vortex dynamics of spin Hamiltonian models
The pattern formation in orientation and ocular dominance columns is one of
the most investigated problems in the brain. From a known cortical structure,
we build spin-like Hamiltonian models with long-range interactions of the
Mexican hat type. These Hamiltonian models allow a coherent interpretation of
the diverse phenomena in the visual map formation with the help of relaxation
dynamics of spin systems. In particular, we explain various phenomena of
self-organization in orientation and ocular dominance map formation including
the pinwheel annihilation and its dependency on the columnar wave vector and
boundary conditions.Comment: 4 pages, 15 figure
Embedding of Cortical Representations by the Superficial Patch System
Pyramidal cells in layers 2 and 3 of the neocortex of many species collectively form a clustered system of lateral axonal projections (the superficial patch system—Lund JS, Angelucci A, Bressloff PC. 2003. Anatomical substrates for functional columns in macaque monkey primary visual cortex. Cereb Cortex. 13:15-24. or daisy architecture—Douglas RJ, Martin KAC. 2004. Neuronal circuits of the neocortex. Annu Rev Neurosci. 27:419-451.), but the function performed by this general feature of the cortical architecture remains obscure. By comparing the spatial configuration of labeled patches with the configuration of responses to drifting grating stimuli, we found the spatial organizations both of the patch system and of the cortical response to be highly conserved between cat and monkey primary visual cortex. More importantly, the configuration of the superficial patch system is directly reflected in the arrangement of function across monkey primary visual cortex. Our results indicate a close relationship between the structure of the superficial patch system and cortical responses encoding a single value across the surface of visual cortex (self-consistent states). This relationship is consistent with the spontaneous emergence of orientation response-like activity patterns during ongoing cortical activity (Kenet T, Bibitchkov D, Tsodyks M, Grinvald A, Arieli A. 2003. Spontaneously emerging cortical representations of visual attributes. Nature. 425:954-956.). We conclude that the superficial patch system is the physical encoding of self-consistent cortical states, and that a set of concurrently labeled patches participate in a network of mutually consistent representations of cortical inpu
Primer to Voltage Imaging With ANNINE Dyes and Two-Photon Microscopy
ANNINE-6 and ANNINE-6plus are voltage-sensitive dyes that when combined with two-photon microscopy are ideal for recording of neuronal voltages in vivo, in both bulk loaded tissue and the dendrites of single neurons. Here, we describe in detail but for a broad audience the voltage sensing mechanism of fast voltage-sensitive dyes, with a focus on ANNINE dyes, and how voltage imaging can be optimized with one-photon and two-photon excitation. Under optimized imaging conditions the key strengths of ANNINE dyes are their high sensitivity (0.5%/mV), neglectable bleaching and phototoxicity, a linear response to membrane potential, and a temporal resolution which is faster than the optical imaging devices currently used in neurobiology (order of nanoseconds). ANNINE dyes in combination with two-photon microscopy allow depth-resolved voltage imaging in bulk loaded tissue to study average membrane voltage oscillations and sensory responses. Alternatively, if ANNINE-6plus is applied internally, supra and sub threshold voltage changes can be recorded from dendrites of single neurons in awake animals. Interestingly, in our experience ANNINE-6plus labeling is impressively stable in vivo, such that voltage imaging from single Purkinje neuron dendrites can be performed for 2 weeks after a single electroporation of the neuron. Finally, to maximize their potential for neuroscience studies, voltage imaging with ANNINE dyes and two-photon microscopy can be combined with electrophysiological recording, calcium imaging, and/or pharmacology, even in awake animals
Depth-resolved microscopy of cortical hemodynamics with optical coherence tomography
We describe depth-resolved microscopy of cortical hemodynamics with high-speed spectral/Fourier domain optical coherence tomography (OCT). Stimulus-evoked changes in blood vessel diameter, flow, and total hemoglobin were measured in the rat somatosensory cortex. The results show OCT measurements of hemodynamic changes during functional activation and represent an important step toward understanding functional hyperemia at the microscopic level.National Institutes of Health (U.S.) (R01-NS057476)National Institutes of Health (U.S.) (P01NS055104)National Institutes of Health (U.S.) (P50NS010828)National Institutes of Health (U.S.) (K99NS067050)National Institutes of Health (U.S.) (R01-CA075289-12)United States. Air Force Office of Scientific Research (FA9550-07-1-0014)United States. Dept. of Defense. Medical Free Electron Laser Program (FA9550-07-1-0101
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