Modulating electrophysiology of motor neural networks via optogenetic stimulation during neurogenesis and synaptogenesis.

Control of electrical activity in neural circuits through network training is a grand challenge for biomedicine and engineering applications. Past efforts have not considered evoking long-term changes in firing patterns of in-vitro networks by introducing training regimens with respect to stages of neural development. Here, we used Channelrhodopsin-2 (ChR2) transfected mouse embryonic stem cell (mESC) derived motor neurons to explore short and long-term programming of neural networks by using optical stimulation implemented during neurogenesis and synaptogenesis. Not only did we see a subsequent increase of neurite extensions and synaptophysin clustering, but by using electrophysiological recording with micro electrode arrays (MEA) we also observed changes in signal frequency spectra, increase of network synchrony, coordinated firing of actions potentials, and enhanced evoked response to stimulation during network formation. Our results demonstrate that optogenetic stimulation during neural differentiation can result in permanent changes that extended to the genetic expression of neurons as demonstrated by RNA Sequencing. To our knowledge, this is the first time that a correlation between training regimens during neurogenesis and synaptogenesis and the resulting plastic responses has been shown in-vitro and traced back to changes in gene expression. This work demonstrates new approaches for training of neural circuits whose electrical activity can be modulated and enhanced, which could lead to improvements in neurodegenerative disease research and engineering of in-vitro multi-cellular living systems.

Dynamic Tracking Algorithm for Time-Varying Neuronal Network Connectivity using Wide-Field Optical Image Video Sequences.

Propagation of signals between neurons and brain regions provides information about the functional properties of neural networks, and thus information transfer. Advances in optical imaging and statistical analyses of acquired optical signals have yielded various metrics for inferring neural connectivity, and hence for mapping signal intercorrelation. However, a single coefficient is traditionally derived to classify the connection strength between two cells, ignoring the fact that neural systems are inherently time-variant systems. To overcome these limitations, we utilized a time-varying Pearson's correlation coefficient, spike-sorting, wavelet transform, and wavelet coherence of calcium transients from DIV 12-15 hippocampal neurons from GCaMP6s mice after applying various concentrations of glutamate. Results provide a comprehensive overview of resulting firing patterns, network connectivity, signal directionality, and network properties. Together, these metrics provide a more comprehensive and robust method of analyzing transient neural signals, and enable future investigations for tracking the effects of different stimuli on network properties.

Optical excitation and detection of neuronal activity.

Optogenetics has emerged as an exciting tool for manipulating neural activity, which in turn, can modulate behavior in live organisms. However, detecting the response to the optical stimulation requires electrophysiology with physical contact or fluorescent imaging at target locations, which is often limited by photobleaching and phototoxicity. In this paper, we show that phase imaging can report the intracellular transport induced by optogenetic stimulation. We developed a multimodal instrument that can both stimulate cells with subcellular spatial resolution and detect optical pathlength (OPL) changes with nanometer scale sensitivity. We found that OPL fluctuations following stimulation are consistent with active organelle transport. Furthermore, the results indicate a broadening in the transport velocity distribution, which is significantly higher in stimulated cells compared to optogenetically inactive cells. It is likely that this label-free, contactless measurement of optogenetic response will provide an enabling approach to neuroscience.

Anomalous heat flow in 8-Pmmn borophene with tilted Dirac cones.

We analytically establish an anomalous transverse flow of heat in 8- Pmmn borophene, one of the several two-dimensional allotropes of Boron. The dispersion of this allotrope contains a pair of anisotropic and tilted Dirac cones which are gapped by placing the 2D B sheet under an intense circularly-polarized illumination. A gap in the Dirac dispersion leads to a finite Berry curvature and connected anomalous Hall effects. In the case of thermoelectrics, this manifests as a heat current perpendicular to the temperature gradient-the thermal Hall effect. A quantitative calculation of the attendant thermal Hall conductivity reveals dependence on the intrinsic anisotropy and tilt of the Dirac cone. Further, by estimating the longitudinal thermal conductivity using the Wiedemann-Franz law, we also outline steps to compute the thermal Hall angle that gauges the generation efficiency of such transverse heat processes. Finally, we touch upon the idea of thermal rectification wherein the direction of flow of the anomalous heat reverses through a simple switch of the polarization of incident light and is of interest in thermal logic circuits.

Spin-dependent magneto-thermopower of narrow-gap lead chalcogenide quantum wells.

A semi-classical analysis of magneto-thermopower behaviour, namely, the Seebeck and Nernst effect (NE) in quantum wells of IV-VI lead salts with significant extrinsic Rashba spin-orbit coupling (RSOC) is performed in this report. In addition to the spin-dependent Seebeck effect that has been observed before, we also theoretically predict a similar spin-delineated behavior for its magneto-thermal analog, the spin-dependent NE. The choice of lead salts follows from a two-fold advantage they offer, in part, to their superior thermoelectric properties, especially PbTe, while their low band gaps and high spin-orbit coupling make them ideal candidates to study RSOC governed effects in nanostructures. The calculations show a larger longitudinal magneto-thermopower for the spin-up electrons while the transverse components are nearly identical. In contrast, for a magnetic field free case, the related power factor calculations reveal a significantly higher contribution from the spin-down ensemble and suffer a reduction with an increase in the electron density. We also discuss qualitatively the limitations of the semi-classical approach for the extreme case of a high magnetic field and allude to the observed thermopower behaviour when the quantum Hall regime is operational. Finally, techniques to modulate the thermopower are briefly outlined.

The electrothermal conductance and heat capacity of black phosphorus.

We study a thermal gradient induced current Ith flow in potassium-doped two-dimensional anisotropic black phosphorus (BP) with semi-Dirac dispersion. The prototype device is a BP channel clamped between two contacts maintained at unequal temperatures. The choice of BP lies in the predicted efficient thermoelectric behaviour. A temperature-induced difference in the Fermi levels of the two contacts drives the current (typified by the electro-thermal conductance) which we calculate using the Landauer transport equation. The current shows an initial rise when the device is operated at lower temperatures. The rise stalls at progressively higher temperatures and Ith acquires a plateau-like flat profile indicating a competing effect between a larger number of transmission modes and a corresponding drop in the Fermi level difference between the contacts. The current is computed for both n- and p-type BP, and the difference thereof is attributed to the particle-hole asymmetry. The utility of such calculations lie in conversion of the heat produced in a miniaturized chip to useful thermopower via a prototypical Seebeck power generator. Unlike the flow of Ith that purportedly utilizes the additional removable heat in a nanoscale device heat, the ability of a material to maintain a steady temperature is reflected in its heat capacity through effective absorption of thermal energy. The heat capacity is formulated in this work for BP via a Sommerfeld expansion. In the concluding part, we draw a microscopic connection between the two seemingly disparate processes of heat removal and absorption by pinning down their origin to the underlying density of states. Finally, a qualitative analysis of a Carnot-like efficiency of the considered thermoelectric engine is performed drawing upon the previous results on thermal current and heat capacity.

Low-temperature thermal transport and thermopower of monolayer transition metal dichalcogenide semiconductors.

We study the low temperature thermal conductivity of single-layer transition metal dichalcogenides (TMDCs). In the low temperature regime where heat is carried primarily through transport of electrons, thermal conductivity is linked to electrical conductivity through the Wiedemann-Franz law (WFL). Using a k.p Hamiltonian that describes the [Formula: see text] and [Formula: see text] valley edges, we compute the zero-frequency electric (Drude) conductivity using the Kubo formula to obtain a numerical estimate for the thermal conductivity. The impurity scattering determined transit time of electrons which enters the Drude expression is evaluated within the self-consistent Born approximation. The analytic expressions derived show that low temperature thermal conductivity (1) is determined by the band gap at the valley edges in monolayer TMDCs and (2) in presence of disorder which can give rise to the variable range hopping regime, there is a distinct reduction. Additionally, we compute the Mott thermopower and demonstrate that under a high frequency light beam, a valley-resolved thermopower can be obtained. A closing summary reviews the implications of results followed by a brief discussion on applicability of the WFL and its breakdown in context of the presented calculations.

Coherent control of an opsin in living brain tissue.

Retinal-based opsins are light-sensitive proteins. The photoisomerization reaction of these proteins has been studied outside cellular environments using ultrashort tailored light pulses1-5. However, how living cell functions can be modulated via opsins by modifying fundamental nonlinear optical properties of light interacting with the retinal chromophore has remained largely unexplored. We report the use of chirped ultrashort near-infrared pulses to modulate light-evoked ionic current from Channelrhodopsin-2 (ChR2) in brain tissue, and consequently the firing pattern of neurons, by manipulating the phase of the spectral components of the light. These results confirm that quantum coherence of the retinal-based protein system, even in a living neuron, can influence its current output, and open up the possibilities of using designer-tailored pulses for controlling molecular dynamics of opsins in living tissue to selectively enhance or suppress neuronal function for adaptive feedback-loop applications in the future.

Optogenetic skeletal muscle-powered adaptive biological machines.

Complex biological systems sense, process, and respond to their surroundings in real time. The ability of such systems to adapt their behavioral response to suit a range of dynamic environmental signals motivates the use of biological materials for other engineering applications. As a step toward forward engineering biological machines (bio-bots) capable of nonnatural functional behaviors, we created a modular light-controlled skeletal muscle-powered bioactuator that can generate up to 300 µN (0.56 kPa) of active tension force in response to a noninvasive optical stimulus. When coupled to a 3D printed flexible bio-bot skeleton, these actuators drive directional locomotion (310 µm/s or 1.3 body lengths/min) and 2D rotational steering (2°/s) in a precisely targeted and controllable manner. The muscle actuators dynamically adapt to their surroundings by adjusting performance in response to "exercise" training stimuli. This demonstration sets the stage for developing multicellular bio-integrated machines and systems for a range of applications.

Scattering times and surface conductivity of Dirac fermions in a 3D topological insulator film with localised impurities.

The zero gap surface states of a 3D-topological insulator host highly mobile Dirac fermions with spin locked to the momentum. The high mobility attributed to the absence of back scattering is reduced in the presence of impurities on the surface. In particular, we discuss and compare scattering times for localised impurities on the surface, scattering between states of opposite helicity located on different surfaces coupled through a hybridisation potential and the role of magnetic impurities. Magnetic impurities give rise to an additional spin suppression factor. The role of warped bands and their influence on topological factors that can enhance the overall surface mobility is examined. Finally, employing a linearised Boltzmann equation approach, surface conductivity calculations for Dirac fermions in a 3D TI is outlined.

The evaluation of non-topological components in Berry phase and momentum relaxation time in a gapped 3D topological insulator.

The zero gap surface states of a 3D-topological insulator host Dirac fermions with spin locked to the momentum. The gap-less Dirac fermions exhibit electronic behaviour different from those predicted in conventional materials. While calculations based on a simple linear dispersion can account for observed experimental patterns, a more accurate description of the physics of these systems and a better agreement between experimental data theoretical results can be obtained by including higher order k terms in the Hamiltonian. In this work, in presence of a time reversal symmetry breaking external magnetic field and higher order warping term, alteration to the topologically ordained Berry phase of (2n + 1)π, momentum relaxation time, and the magneto-conductivity tensors is established. The relation between scattering times and the deviations to topological Berry phase of π is also emphasized.

Tumor necrosis factor enhances the sleep-like state and electrical stimulation induces a wake-like state in co-cultures of neurons and glia.

We characterise sleep-like states in cultured neurons and glia during development in vitro as well as after electrical stimulation, the addition of tumor necrosis factor alpha (TNF), and the combination of TNF plus electrical stimulation. We also characterise optogenetic stimulation-induced ATP release and neuronal interleukin-1 and TNF expression in vitro demonstrating the activity dependence of these putative sleep-regulatory substances. Action potential (AP) burstiness, expressed as the burstiness index (BI), synchronization of slow electrical potentials between recording electrodes (SYN), and slow wave (SW) power (0.25-3.75 Hz) determined using fast Fourier analyses emerged as network properties, maturing after 2 weeks in culture. Homologous in vivo measures are used to characterise sleep. Electrical stimulation reduced the BI, SYN and SW power values during and/or after the stimulus period. One day later, homeostasis was evident from rebounds of SYN and SW power values to above baseline levels; the magnitude of the rebound was stimulus pattern-dependent. The addition of TNF enhanced BI, SYN and SW power values, suggesting the induction of a deeper sleep-like state. Electrical stimulation reversed these TNF effects, suggesting the network state was more wake-like. The day after TNF plus electrical stimulation, the changes in SYN and SW power values were dependent upon the stimulus patterns the cells received the day before. We conclude that sleep and wake states in cultured in vitro networks can be controlled and they share molecular regulatory mechanisms with local in vivo networks. Further, sleep is an activity-dependent emergent local network property.

On the stability of the soluble amyloid aggregates.

Many amyloid proteins form metastable soluble aggregates (or protofibrils, or protein nanoparticles, with characteristic sizes from approximately 10 to a few hundred nm). These can coexist with protein monomers and amyloid precipitates. These soluble aggregates are key determinants of the toxicity of these proteins. It is therefore imperative to understand the physical basis underlying their stability. Simple nucleation theory, typically applied to explain the kinetics of amyloid precipitation, fails to predict such intermediate stable states. We examine stable nanoparticles formed by the Alzheimer's amyloid-beta peptide (40 and 42 residues), and by the protein barstar. These molecules have different hydrophobicities, and therefore have different short-range attractive interactions between the molecules. We also vary the pH and the ionic strength of the solution to tune the long-range electrostatic repulsion between them. In all the cases, we find that increased long-range repulsion results in smaller stable nanoparticles, whereas increased hydrophobicity produces the opposite result. Our results agree with a charged-colloid type of model for these particles, which asserts that growth-arrested colloid particles can result from a competition between short-range attraction and long-range repulsion. The nanoparticle size varies superlinearly with the ionic strength, possibly indicating a transition from an isotropic to a linear mode of growth. Our results provide a framework for understanding the stability and growth of toxic amyloid nanoparticles, and provide cues for designing effective destabilizing agents.

EGFR juxtamembrane domain, membranes, and calmodulin: kinetics of their interaction.

Calcium/calmodulin (Ca/CaM) binds to the intracellular juxtamembrane domain (JMD) of the epidermal growth factor receptor (EGFR). The basic JMD also binds to acidic lipids in the inner leaflet of the plasma membrane, and this interaction may contribute an extra level of autoinhibition to the receptor. Binding of a ligand to the EGFR produces a rapid increase in intracellular calcium, [Ca2+]i, and thus Ca/CaM. How does Ca/CaM compete with the plasma membrane for the JMD? Does Ca/CaM directly pull the JMD off the membrane or does Ca/CaM only bind to the JMD after it has dissociated spontaneously from the bilayer? To answer this question, we studied the effect of Ca/CaM on the rate of dissociation of fluorescent JMD peptides from phospholipid vesicles by making kinetic stop-flow measurements. Ca/CaM increases the rate of dissociation: an analysis of the differential equations that describe the dissociation shows that Ca/CaM must directly pull the basic JMD peptide off the membrane surface. These measurements lead to a detailed atomic-level mechanism for EGFR activation that reconciles the existence of preformed EGFR dimers/oligomers with the Kuriyan allosteric model for activation of the EGFR kinase domains.

Evidence for a second, high affinity Gbetagamma binding site on Galphai1(GDP) subunits.

It is well known that Galpha(i1)(GDP) binds strongly to Gbetagamma subunits to form the Galpha(i1)(GDP)-Gbetagamma heterotrimer, and that activation to Galpha(i1)(GTP) results in conformational changes that reduces its affinity for Gbetagamma subunits. Previous studies of G protein subunit interactions have used stoichiometric amounts of the proteins. Here, we have found that Galpha(i1)(GDP) can bind a second Gbetagamma subunit with an affinity only 10-fold weaker than the primary site and close to the affinity between activated Galpha(i1) and Gbetagamma subunits. Also, we find that phospholipase Cbeta2, an effector of Gbetagamma, does not compete with the second binding site implying that effectors can be bound to the Galpha(i1)(GDP)-(Gbetagamma)(2) complex. Biophysical measurements and molecular docking studies suggest that this second site is distant from the primary one. A synthetic peptide having a sequence identical to the putative second binding site on Galpha(i1) competes with binding of the second Gbetagamma subunit. Injection of this peptide into cultured cells expressing eYFP-Galpha(i1)(GDP) and eCFP-Gbetagamma reduces the overall association of the subunits suggesting this site is operative in cells. We propose that this second binding site serves to promote and stabilize G protein subunit interactions in the presence of competing cellular proteins.

Caveolin-1 alters Ca(2+) signal duration through specific interaction with the G alpha q family of G proteins.

Caveolae are membrane domains having caveolin-1 (Cav1) as their main structural component. Here, we determined whether Cav1 affects Ca(2+) signaling through the Galpha(q)-phospholipase-Cbeta (PLCbeta) pathway using Fischer rat thyroid cells that lack Cav1 (FRTcav(-)) and a sister line that forms caveolae-like domains due to stable transfection with Cav1 (FRTcav(+)). In the resting state, we found that eCFP-Gbetagamma and Galpha(q)-eYFP are similarly associated in both cell lines by Forster resonance energy transfer (FRET). Upon stimulation, the amount of FRET between Galpha(q)-eYFP and eCFP-Gbetagamma remains high in FRTcav(-) cells, but decreases almost completely in FRTcav(+) cells, suggesting that Cav1 is increasing the separation between Galpha(q)-Gbetagamma subunits. In FRTcav(-) cells overexpressing PLCbeta, a rapid recovery of Ca(2+) is observed after stimulation. However, FRTcav(+) cells show a sustained level of elevated Ca(2+). FRET and colocalization show specific interactions between Galpha(q) and Cav1 that increase upon stimulation. Fluorescence correlation spectroscopy studies show that the mobility of Galpha(q)-eGFP is unaffected by activation in either cell type. The mobility of eGFP-Gbetagamma remains slow in FRTcav(-) cells but increases in FRTcav(+) cells. Together, our data suggest that, upon stimulation, Galpha(q)(GTP) switches from having strong interactions with Gbetagamma to Cav1, thereby releasing Gbetagamma. This prolongs the recombination time for the heterotrimer, thus causing a sustained Ca(2+) signal.

Signaling through a G Protein-coupled receptor and its corresponding G protein follows a stoichiometrically limited model.

The bradykinin receptor is a G protein-coupled receptor (GPCR) that is coupled to the Galpha(q) family of heterotrimeric G proteins. In general, a GPCR can exert intracellular signals either by transiently associating with multiple diffusing G protein subunits or by activating a G protein that is stably bound to the receptor, thus generating a signal that is limited by the stoichiometry of the complex. Here we have distinguished between these models by monitoring the association of type 2 bradykinin receptor (B(2)R) and the Galpha(q)/Gbetagamma heterotrimer in living human embryonic kidney 293 cells expressing fluorescent-tagged proteins. Stable B(2)R-Galpha(q) x Gbetagamma complexes are observed in resting cells by fluorescence resonance energy transfer from either Galpha(q)-eCFP or eCFP-Gbetagamma to B(2)R-eYFP. Stimulating the cells with bradykinin causes detachment of B(2)R from the G protein subunits as the receptor internalizes into early endosomes, with a corresponding elimination of B(2)R-G protein fluorescence resonance energy transfer because Galpha(q) and its associated Gbetagamma remain on the plasma membrane. Single point and scanning fluorescence correlation spectroscopy measurements show that a portion of B(2)R molecules diffuses with a mobility corresponding to dimers or small oligomers, whereas a second fraction diffuses in higher order molecular assemblies. Our studies support a model in which receptors are pre-coupled with their corresponding G proteins in the basal state of cells thereby limiting the response to an external signal to a defined stoichiometry that allows for a rapid and directed cellular response.

Membrane-permeable calmodulin inhibitors (e.g. W-7/W-13) bind to membranes, changing the electrostatic surface potential: dual effect of W-13 on epidermal growth factor receptor activation.

Membrane-permeable calmodulin inhibitors, such as the napthalenesulfonamide derivatives W-7/W-13, trifluoperazine, and calmidazolium, are used widely to investigate the role of calcium/calmodulin (Ca2+/CaM) in living cells. If two chemically different inhibitors (e.g. W-7 and trifluoperazine) produce similar effects, investigators often assume the effects are due to CaM inhibition. Zeta potential measurements, however, show that these amphipathic weak bases bind to phospholipid vesicles at the same concentrations as they inhibit Ca2+/CaM; this suggests that they also bind to the inner leaflet of the plasma membrane, reducing its negative electrostatic surface potential. This change will cause electrostatically bound clusters of basic residues on peripheral (e.g. Src and K-Ras4B) and integral (e.g. epidermal growth factor receptor (EGFR)) proteins to translocate from the membrane to the cytoplasm. We measured inhibitor-mediated translocation of a simple basic peptide corresponding to the calmodulin-binding juxtamembrane region of the EGFR on model membranes; W-7/W-13 causes translocation of this peptide from membrane to solution, suggesting that caution must be exercised when interpreting the results obtained with these inhibitors in living cells. We present evidence that they exert dual effects on autophosphorylation of EGFR; W-13 inhibits epidermal growth factor-dependent EGFR autophosphorylation under different experimental conditions, but in the absence of epidermal growth factor, W-13 stimulates autophosphorylation of the receptor in four different cell types. Our interpretation is that the former effect is due to W-13 inhibition of Ca2+/CaM, but the latter results could be due to binding of W-13 to the plasma membrane.

Measuring fast dynamics in solutions and cells with a laser scanning microscope.

Single-point fluorescence correlation spectroscopy (FCS) allows measurements of fast diffusion and dynamic processes in the microsecond-to-millisecond time range. For measurements on living cells, image correlation spectroscopy (ICS) and temporal ICS extend the FCS approach to diffusion times as long as seconds to minutes and simultaneously provide spatially resolved dynamic information. However, ICS is limited to very slow dynamics due to the frame acquisition rate. Here we develop novel extensions to ICS that probe spatial correlations in previously inaccessible temporal windows. We show that using standard laser confocal imaging techniques (raster-scan mode) not only can we reach the temporal scales of single-point FCS, but also have the advantages of ICS in providing spatial information. This novel method, called raster image correlation spectroscopy (RICS), rapidly measures during the scan many focal points within the cell providing the same concentration and dynamic information of FCS as well as information on the spatial correlation between points along the scanning path. Longer time dynamics are recovered from the information in successive lines and frames. We exploit the hidden time structure of the scan method in which adjacent pixels are a few microseconds apart thereby accurately measuring dynamic processes such as molecular diffusion in the microseconds-to-seconds timescale. In conjunction with simulated data, we show that a wide range of diffusion coefficients and concentrations can be measured by RICS. We used RICS to determine for the first time spatially resolved diffusions of paxillin-EGFP stably expressed in CHOK1 cells. This new type of data analysis has a broad application in biology and it provides a powerful tool for measuring fast as well as slower dynamic processes in cellular systems using any standard laser confocal microscope.

Fluctuation correlation spectroscopy with a laser-scanning microscope: exploiting the hidden time structure.

Images obtained with a laser-scanning microscope contain a time structure that can be exploited to measure fast dynamics of molecules in solution and in cells. The spatial correlation approach provides a simple algorithm to extract this information. We describe the analysis used to process laser-scanning images of solutions and cells to obtain molecular diffusion constant in the microsecond to second timescale.