Bridging Quantum Chemical Predictions of Excitation Energies with Experimental Data for Voltage-Sensitive Fluorescent Dyes.

Voltage sensitive dye (VSD) imaging of multiple neurons becomes one of the most promising up-to-date methods to investigate neuronal network activity. However, optical imaging signals are often superimposed by noise and artifacts. Hence, post-processing methods are needed to overcome this corruption and separate neuronal activity from other signals. One of the significant artifacts in VSD imaging is bleaching, a decrease of the optical signal while the recorded signal of the local field potentials remains unchanged [1]. In this study we used independent component analysis (ICA) in comparison to principal component analysis (PCA) and detrend method to separate the neuronal VSD signals from bleaching artifacts. ICA is a blind source separation method that has been used in many different approaches such as to recover action potentials of neurons in multiple-detector optical recordings [3]. We used the ICA-DTU Toolbox [http://www2.imm.dtu.dk/pubdb/views/publication_details.php?id=4043] with maximum likelihood formulation to identify neuronal signals and the effect of bleaching. Experimentally, we recorded from Retzius cells of an isolated midbody ganglion of the electrophysiologically well- characterized leech nervous system. VSD imaging (Figure A) based on a new VSD dye with a very good signal-to-noise ratio [2] was combined with simultaneous intracellular recording of the membrane potential (Figure B) and electrical stimulation. VSD signals obtained from the region of the Retzius cell body (Figure ​(Figure1A1A and ​and1C,1C, red) and from a slightly larger region (Figure ​(Figure1A1A and ​and1C,1C, blue) were used as input signals to ICA. Of the two components returned by the algorithm one decreased exponentially, while the other resembled neuronal activity. Figure ​Figure1D1D shows an exponential fit of the component representing bleaching (green), and the estimated neuronal activity (red). Taking the lower sampling rate of the VSD signal (100 Hz vs 10,000 Hz) into account, this estimate reflects the dynamics of the intracellular recording (1B) well. While PCA was not able to separate bleaching from neuronal signals (not shown), the detrend method also yielded good results (Figure ​(Figure1E).1E). This method fits a piecewise linear line (1E, green) to the VSD signal, representing bleaching effect. However, the difference (1E, red) between the original trace and this line represents the graded de-and hyperpolarisations of the membrane potential seen in the intracellular recording (1B) less well than the ICA result (1D, red). Figure 1 VSD and intracellular recording of leech ganglion and analysis results.

Optical recording of membrane potential permits spatially resolved measurement of electrical activity in subcellular regions of single cells, which would be inaccessible to electrodes, and imaging of spatiotemporal patterns of action potential propagation in excitable tissues, such as the brain or heart. However, the available voltage-sensitive dyes (VSDs) are not always spectrally compatible with newly available optical technologies for sensing or manipulating the physiological state of a system. Here, we describe a series of 19 fluorinated VSDs based on the hemicyanine class of chromophores. Strategic placement of the fluorine atoms on the chromophores can result in either blue or red shifts in the absorbance and emission spectra. The range of one-photon excitation wavelengths afforded by these new VSDs spans 440-670 nm; the two-photon excitation range is 900-1,340 nm. The emission of each VSD is shifted by at least 100 nm to the red of its one-photon excitation spectrum. The set of VSDs, thus, affords an extended toolkit for optical recording to match a broad range of experimental requirements. We show the sensitivity to voltage and the photostability of the new VSDs in a series of experimental preparations ranging in scale from single dendritic spines to whole heart. Among the advances shown in these applications are simultaneous recording of voltage and calcium in single dendritic spines and optical electrophysiology recordings using two-photon excitation above 1,100 nm.

Simultaneous Neuronal Activity Measurement Using a Microelectrode Array Recording and Voltage Sensitive Dye Imaging

Optical monitoring of synaptic transmission in bullfrog sympathetic ganglia using a voltage-sensitive dye

The hydroxylation reaction catalyzed by p-hydroxybenzoate hydroxylase and the Baeyer-Villiger reaction catalyzed by cyclohexanone monooxygenase are investigated by means of quantum mechanical/molecular mechanical (QM/MM) calculations at different levels of QM theory. The geometries of the stationary points along the reaction profile are obtained from QM/MM geometry optimizations, in which the QM region is treated by density functional theory (DFT). Relative energies are determined from single-point QM/MM calculations using the domain-based local pair natural orbital coupled cluster DLPNO-CCSD(T) method as QM component. The results are compared with single-point DFT/MM energies obtained using popular density functionals and with available experimental and computational data. It is found that the choice of the QM method strongly affects the computed energy profiles for these reactions. Different density functionals provide qualitatively different energy barriers (variations of the order of 10 kcal/mol in both reactions), thus limiting the confidence in DFT/MM computational predictions of energy profiles. On the other hand, the use of the DLPNO-CCSD(T) method in conjunction with large QM regions and basis sets makes it possible to achieve high accuracy. A critical discussion of all the technical aspects of the calculations is given with the aim of aiding computational chemists in the application of the DLPNO-CCSD(T) methodology in QM/MM calculations.

Neuron electrical activity was recorded using an optical method in which the probes were widely used monomolecular voltage-sensitive dyes. Current methods of loading voltage-sensitive dyes into nervous tissue have serious limitations, severely restricting their areas of use (insolubility in water, the need for toxic organic solvents, and the toxicity of the voltage-sensitive dyes themselves at high concentrations). We have developed a new method of loading voltage-sensitive dyes by “shooting” dye-coated gold microparticles into living brain slices. Three-dimensional reconstruction of nervous tissue fluorescence with a scanning confocal microscope showed that after loading with dye by this method, it propagated along the cell membrane, completely staining only one target cell with its processes, without spreading to surrounding cells via the intercellular fluid or through cell contacts. This method of staining neurons can be used for the optical recording of the electrical activity of individual neurons and to analyze the distribution of electric voltages across the compartments of excitable cells (axons and dendrites).

Optical imaging of neuronal activity.

Standing Waves and Traveling Waves Distinguish Two Circuits in Visual Cortex

Using an optical imaging technique with voltage-sensitive dyes (VSDs), we investigated the functional organization and architecture of the central nervous system (CNS) during embryogenesis. In the embryonic nervous system, a merocyanine-rhodanine dye, NK2761, has proved to be the most useful absorption dye for detecting neuronal activity because of its high signal-to-noise ratio (S/N), low toxicity and small dye bleaching. In the present study, we evaluated the suitability of fluorescence VSDs for optical recording in the embryonic CNS. We screened eight styryl (hemicyanine) dyes in isolated brainstem-spinal cord preparations from 7-day-old chick embryos. Measurements of voltage-related optical signals were made using a multiple-site optical recording system. The signal size, S/N, photobleaching, effects of perfusion and recovery of neural responses after staining were compared. We also evaluated optical responses with various magnifications. Although the S/N was lower than with the absorption dye, clear optical responses were detected with several fluorescence dyes, including di-2-ANEPEQ, di-4-ANEPPS, di-3-ANEPPDHQ, di-4-AN(F)EPPTEA, di-2-AN(F)EPPTEA and di-2-ANEPPTEA. Di-2-ANEPEQ showed the largest S/N, whereas its photobleaching was faster and the recovery of neural responses after staining was slower. Di-4-ANEPPS and di-3-ANEPPDHQ also exhibited a large S/N but required a relatively long time for recovery of neural activity. Di-4-AN(F)EPPTEA, di-2-AN(F)EPPTEA and di-2-ANEPPTEA showed smaller S/Ns than di-2-ANEPEQ, di-4-ANEPPS and di-3-ANEPPDHQ; but the recovery of neural responses after staining was faster. This study demonstrates the potential utility of these styryl dyes in optical monitoring of voltage changes in the embryonic CNS.

Event Abstract Back to Event Separation of single neurons from optical recordings in Tritonia diomedea using ICA Optical recordings obtained with photodiode arrays and voltage-sensitive dyes allow firing of large numbers of neurons to be recorded during behaviorally-relevant motor programs. Although this technique has tremendous potential for network studies, the resulting datasets can be challenging to interpret; one detector may record multiple neurons and one neuron may appear on many detectors. Earlier studies applied independent component analysis (ICA), a blind source separation technique, to optical traces from Tritonia diomedea's escape swim behavior, to recover components from individual neurons [1] and identify unreported neurons involved in the behavior [2]. Using intracellular and optical methods [3], we evaluate the accuracy of neuronal activity that ICA returns and demonstrate an application of these methods, measuring the change in individual neurons’ activity following stimulus adaptation. To evaluate accuracy, we recorded from neurons in Tritonia with intracellular electrodes while imaging with a 464-element photodiode array using the fast voltage-sensitive absorbance dye RH-155. ICA was run on the optical traces, transforming them into statistically independent components. ICA returned one component for neurons detected by multiple diodes and separated neurons mixed on a single diode. Intracellular recordings confirmed the accuracy of this technique. For each intracellular recording, we found a component that corresponded exactly. Also, the location returned by ICA matched that of the electrode. Additionally, these methods allow us to drive an identified neuron and image its functional connectivity to large numbers of other individual neurons, before and after a treatment of interest. We imaged the responses of several pedal ganglion neurons to a test train of action potentials in CPG neuron C2 driven with an intracellular electrode. Then we applied a sensitizing nerve shock stimulus. Two minutes later we imaged the responses of the same neurons to a second test train. Combining the records and applying ICA, we observed how responses of individual neurons changed after the sensitizing stimulus. Optical recordings in combination with ICA allow quick identification of functional synaptic connections onto previously unknown neurons. Using location maps from ICA, those neurons can be penetrated with intracellular electrodes within the same experiment, enabling new experiments to probe the Tritonia brain over a wide range of conditions and time scales. Given our intracellular validation of ICA's unmixing, these methods promise to be a powerful tool for discovery of unreported neurons and studies of functional connectivity and network plasticity.

Use of voltage-sensitive dyes and optical recordings in the central nervous system

The development of modern neuroscience tools has a significant impact on the progress in the field of neuroscience (Kandel, 1982). New tools has greatly faciliated the neuroscience resreach, and can be critical for studies of brain circuit organization and function. Although many approaches are useful by themselves, it is desirable to combine existing powerful techniques to harness each technique's advantages and compensate for limitations. In many brain areas, neuronal circuits are segregated into anatomically discrete areas such as specific lamina and modules or compartments (Mountcastle, 1997). Functional imaging of these brain areas is particularly useful in characterizing circuit properties. Fast voltagesensitive dye (VSD) imaging, which detects neuronal membrane potential changes via shifts in the dye absorption /or fluorescence emission in response to varying membrane potentials, offers a great means of simultaneous monitoring neuronal activities from many locations with high spatial and temporal resolutions. With new dyes and modern imaging apparatus, VSD imaging has been widely used to study spatiotemporal dynamics of population neuronal activity in cortical tissue both in vivo and in vitro (Grinvald & Hildesheim, 2004). Particularly, for in vitro brain slices, fast VSD imaging is important for mapping circuit organization and response dynamics, and more recently has been used to probe functional abnormalities in models of neurological and psychiatric disorders (Ang et al., 2006; Airan et al., 2007). One major limitation of most in vitro VSD imaging studies, however, lies in that the imaged neuronal responses are either spontaneous seizure activities through pharmacological manipulations or induced by electric stimulations (Petersen & Sakmann, 2001; Huang et al., 2004; Ang et al., 2006). Significant disadvantages of electric stimulation include indiscriminate activation of axons of passage, slow and inefficient placement of multiple stimulation locations, and tissue damage. In comparison, optical stimulation including laser scanning photostimulation (LSPS) either by glutamate uncaging or direct activation of light-sensitive channels (e.g., channelrhodopsin-2) enables rapid and noninvasive photoactivation of neurons with great convenience and superior spatial resolution in practical experiments (Callaway & Katz, 1993; Boyden et al., 2005; Petreanu et al., 2009). Combining whole-cell recordings from single neurons with photostimulation of clusters of presynaptic neurons permits extensive mapping of local functional inputs to individually recorded neurons (Schubert et al., 2003; Shepherd & Svoboda, 2005; Xu & Callaway, 2009).

Due to the higher computational cost relative to pure molecular mechanical (MM) simulations, hybrid quantum mechanical/molecular mechanical (QM/MM) free energy simulations particularly require a careful consideration of balancing computational cost and accuracy. Here, we review several recent developments in free energy methods most relevant to QM/MM simulations and discuss several topics motivated by these developments using simple but informative examples that involve processes in water. For chemical reactions, we highlight the value of invoking enhanced sampling technique (e.g. replica-exchange) in umbrella sampling calculations and the value of including collective environmental variables (e.g. hydration level) in metadynamics simulations; we also illustrate the sensitivity of string calculations, especially free energy along the path, to various parameters in the computation. Alchemical free energy simulations with a specific thermodynamic cycle are used to probe the effect of including the first solvation shell into the QM region when computing solvation free energies. For cases where high-level QM/MM potential functions are needed, we analyse two different approaches: the QM/MM–MFEP method of Yang and co-workers and perturbative correction to low-level QM/MM free energy results. For the examples analysed here, both approaches seem productive although care needs to be exercised when analysing the perturbative corrections.

Voltage-sensitive dye imaging of population neuronal activity in cortical tissue

High-density cultured neuronal networks have been used to evaluate synchronized features of neuronal populations. Voltage-sensitive dye (VSD) imaging of a dissociated cultured neuronal network is a critical method for studying synchronized neuronal activity in single cells. However, the signals of VSD are generally too faint-that is, the signal-to-noise ratio (S/N) is too low-to detect neuronal activity. In our previous research, a silver (Ag) plasmonic chip enhanced the fluorescence intensity of VSD to detect spontaneous neural spikes on VSD imaging. However, no high-density network was cultivated on the Ag plasmonic chip, perhaps because of the chemical instability of the Ag surface. In this study, to overcome the instability of the chip, we used a chemically stable gold (Au) plasmonic dish, which was a plastic dish with a plasmonic chip pasted to the bottom, to observe neuronal activity in a high-density neuronal network. We expected that the S/N in real-time VSD imaging of the Au plasmonic chip would be improved compared to that of a conventional glass-bottomed dish, and we also expected to detect frequent neural spikes. The increase in the number of spikes when inhibitory neurotransmitter receptors were inhibited suggests that the spikes corresponded to neural activity. Therefore, real-time VSD imaging of an Au plasmonic dish was effective for measuring spontaneous network activity in a high-density neuronal network at the spatial resolution of a single cell.