Parijat Sengupta | University of Illinois at Urbana-Champaign (original) (raw)

Papers by Parijat Sengupta

Research paper thumbnail of Regulation of G Protein Signals by Membrane Domains

Research paper thumbnail of 1 Biophysical Journal, Volume 96 Supporting Material EGFR Juxtamembrane Domain, Membranes and Calmodulin: Kinetics of Their Interaction

bound to phospholipid vesicles Ca2+/CaM rips the fluorescent peptide from the phospholipid vesicl... more bound to phospholipid vesicles Ca2+/CaM rips the fluorescent peptide from the phospholipid vesicles Mixing chamber FIGURE S1 Cartoon illustrating the stopped flow experiments designed to measure the effect of Ca/CaM on the rate of peptide dissociation from vesicles. Acrylodan-labeled EGFR(645-660) peptides bound to phospholipid vesicles are in one syringe and calcium/calmodulin is in the other syringe. The acrylodan fluorescence is quenched when the peptide is bound to the membrane, which contains NBD-PS. Upon mixing the solutions together rapidly, calcium/calmodulin rips the peptide from the surface and allows the fluorescence to increase, which is followed as a function of time. The dead time of the apparatus is ~ 5 ms. Peptide labeling and synthesis. To attach the acyrlodan label to EGFR(645-660), 1mg of peptide was dissolved in 0.8mL 10mM K2HPO4/KH2PO4, pH 7 buffer, mixed with the probe dissolved in DMF (1:1 probe:peptide molar ratio) and incubated for at least 1h. Labeled pepti...

Research paper thumbnail of Optogenetic skeletal muscle-powered adaptive biological machines

Proceedings of the National Academy of Sciences, 2016

Significance Understanding the design rules that govern the structure and function of natural bio... more Significance Understanding the design rules that govern the structure and function of natural biological systems gives us the ability to forward engineer machines integrated with and powered by biological components. Such machines, or “bio-bots,” can sense, process, and respond to dynamic environmental signals in real time, enabling a variety of applications. Here we present a modular optogenetic muscle actuator used to power actuation and locomotion of 3D printed flexible skeletons. Observing and controlling the functional response of such muscle-powered machines helps replicate the complex adaptive functionality we observe in natural biological systems. This demonstration thus sets the stage for building the next generation of bio-integrated machines and systems targeted at a diverse array of functional tasks.

Research paper thumbnail of Optogenetic Stimulation of Channelrhodopsin-2 Expressing Neurons Flips Cortical Networks from Low to High Activity State

Biophysical Journal, 2014

Research paper thumbnail of Role of caveolae in the duration of g protein signals

Research paper thumbnail of Electrical properties and the surface characteristics of lanthanum oxide/water interface

Journal of Colloid and Interface Science, 1988

Etude de l'adsorption des ions OH − et H + sur une electrode de verre recouverte d'oxyde ... more Etude de l'adsorption des ions OH − et H + sur une electrode de verre recouverte d'oxyde de lanthane (III), en fonction du pH et de la force ionique de la solution

Research paper thumbnail of Signaling through a G Protein-coupled Receptor and Its Corresponding G Protein Follows a Stoichiometrically Limited Model

Journal of Biological Chemistry, 2007

Research paper thumbnail of EGFR Juxtamembrane Domain, Membranes, and Calmodulin: Kinetics of Their Interaction

Biophysical Journal, 2009

Calcium/calmodulin (Ca/CaM) binds to the intracellular juxtamembrane domain (JMD) of the epiderma... more 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, [Ca 2þ ] 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.

Research paper thumbnail of The ATP-cytokine-adenosine hypothesis: How the brain translates past activity into sleep

Sleep and Biological Rhythms, 2011

Tumor necrosis factor alpha (TNF), interleukin-1 beta (IL1), and other cytokines are involved in ... more Tumor necrosis factor alpha (TNF), interleukin-1 beta (IL1), and other cytokines are involved in non-rapid eye movement sleep (NREM) regulation under physiological and inflammatory conditions. Brain levels of IL1 and TNF increase with prolonged wakefulness. Injection of exogenous IL1 or TNF, mimicking sleep loss, induces sleepiness, excess sleep, fatigue, poor cognition, and enhanced sensitivity to pain. These symptoms characterize the syndrome associated with sleep loss. Extracellular ATP released during neuro-and glio-transmission, acting via purine P2 receptors on glia, releases IL1 and TNF. This extracellular ATP mechanism may provide an index of activity used by the brain to keep track of prior wakefulness. Prolonged wakefulness is associated with enhanced neuronal activity. TNF and IL1, in turn, act on neurons to change their intrinsic properties and sensitivities to neurotransmitters and neuromodulators such as adenosine and glutamate. Such actions change network input-output properties (i.e. state shift). State oscillations, for instance, occur within cortical columns and are responsive to TNF. Sleep is thus viewed as a local usedependent process regulated in part by cytokines. Further, state oscillations are viewed as a fundamental process of any neuronal/glia network. To investigate these hypotheses we developed an in vitro neuronal/glia culture system exhibiting field potential oscillations and have mathematically modeled the local use-dependent view of sleep initiation. These views have profound implications for sleep pathologies and function.

Research paper thumbnail of Optogenetic Stimulation Enhances Interleukin-1beta Levels in Cultured Neurons

Biophysical Journal, 2011

recordings now reliably deliver simultaneous signals from a hundred or more neurons or networks. ... more recordings now reliably deliver simultaneous signals from a hundred or more neurons or networks. However, many analytic techniques are presently computationally limited to smaller numbers of signals, severely limiting our ability to relate these neural signals to brain functions including sensation, perception, decision, and action. To address this imbalance, we are developing a new open source Neurophysiology Extended Analysis Tool. NEAT leverages existing code bases and new massively-parallel computational technology to enable any multielectrode lab to perform high-throughput informative analyses. We begin with linked evaluations of: 1) computational bottlenecks in analytic routines, many information-theoretic, developed for our Spike Train Analysis Toolkit and distributed via neuroanalysis.org to over 1,600 sites [Goldberg et al. Neuroinformatics 7, 165-178, 2009], and 2) specific capabilities and restrictions of new graphics-processor-derived computational engines supplied on inexpensive drop-in cards. We project a greater than order of magnitude speedup that will allow many offline analyses to be performed in real time during experiments, and now-impractical questions to be explored offline in reasonable compute times. For example, pairwise analyses now possible on 10 or fewer neurons may be extendible to 50 or more. We focus on information-theoretic measures and standard pairwise correlations, JPSTHs, spectra, coherences, and new and significant analyses that present significant loads for multineuron recordings. To aid communities planning similar GPU-enabled analyses, we note that the complex, structured, and hierarchic GPU architecture requires special optimization strategies: decomposing code into >1,000 simultaneous threads is needed to efficiently use the 448 cores on new GPUs, data should be loaded into on-chip memory once and re-used, avoiding transfers to other memory layers, kernel processes must optimize thread/kernel and thread/block instruction execution in few clock cycles, flow control code should control multi-thread warps, not individual threads. Support: MH057153/MH068012.

Research paper thumbnail of Computational algorithm for assessing inter-neuronal connectivity to optimize optogenetic stimulation and neural circuit activity (Conference Presentation)

Optogenetics and Optical Manipulation 2018, 2018

All-optical systems for stimulating and imaging neuronal activity have served as powerful tools f... more All-optical systems for stimulating and imaging neuronal activity have served as powerful tools for understanding the underlying circuitry of the brain. Experiments using these setups, however, tend to choose stimulation locations based solely on what brain regions are of interest, and take for granted that stimulation effects may vary even within localized brain regions. We thus have developed an algorithm for acquiring neuronal activity via calcium imaging data to assess network connectivity. These parameters include the signal rise time, decay time, inter-event intervals, and the timing and amplitude of signal peaks. These parameters are then compared between cell clusters for similarities, and used as a basis for establishing interconnectivity. Additionally, we have incorporated both temporal and spatial correlation functions to assess inter-neuronal connectivity based on these parameters. This data is then run through a genetic algorithm, applying weights to cells with similar parameters to learn which are interconnected in a given field-of-view. For this study, hippocampal neurons extracted from 2 day old transgenic mice (GCaMP6s, Jackson Labs), - cultured for 2 weeks and imaged under single and two-photon conditions. Single-photon imaging was performed under a commercial Zeiss microscope, whereas two-photon imaging was performed with an in-house imaging system. Results demonstrate a strong correlation between these parameters and cellular connectivity, making them noteworthy markers for targeted stimulation. This study demonstrates an efficient method of assessing network connectivity for various imaging techniques, and hence directed targeting for optogenetic stimulation.

Research paper thumbnail of Reconstruction and depixelation of multicore fiber bundle images by fiber bundle rotation (Conference Presentation)

Multicore fiber bundles for imaging and stimulating optogenetically modified neurons have been la... more Multicore fiber bundles for imaging and stimulating optogenetically modified neurons have been largely adopted in neurophotonics research. They allow for directed, single-cell stimulation and imaging of neuronal activity. An inherent limitation of these bundles is the presence and detection of the empty space between individual fibers, resulting in a loss of significant amounts of data, and reduced image quality due to pixilation effects. We propose a novel approach and algorithm to depixelation and image reconstruction from fiber bundles that utilizes multiple image frames collected during on-axis fiber bundle rotation. The approach involves first acquiring the Fourier transform of a stationary, unrotated image, followed by its rotated counterparts. The phase information from each image is then acquired, cross-correlated, and the angle of rotation determined from this correlation. Rotated images are then weighed and summed to generate a final reconstructed, depixelated image. Simul...

Research paper thumbnail of Optical excitation and detection of neuronal activity

Journal of biophotonics, Jan 12, 2018

Optogenetics has emerged as an exciting tool for manipulating neural activity, which in turn, can... more 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 changes with nanometer scale sensitivity. We found that optical pathlength 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...

Research paper thumbnail of Spatial control of in vivo optogenetic light stimulation and recording via an imaging fiber bundle (Conference Presentation)

Optogenetics and Optical Manipulation, 2017

Light delivery in in vivo optogenetic applications are typically accomplished via a single multim... more Light delivery in in vivo optogenetic applications are typically accomplished via a single multimode fiber that diffuses light over a large area of the brain, and relies heavily on the spatial distribution of transfected light-sensitive neurons for targeted control. In our investigations, an imaging fiber bundle (Schott, 1534702) containing 4,500 individual fibers, each with a diameter of 7.5 µm, and an overall outer bundle diameter of 530 µm, was used as the conduit for light delivery and optical recording/imaging in neuron cultures and in in vivo mouse brain. We demonstrated that the use of this fiber bundle, in contrast to a single multimode fiber, allowed for individually-addressable fibers, spatial selectivity at the stimulus site, precise control of light delivery, and full field-of-view imaging and/or optical recordings of neurons. An objective coupled the two continuous wave diode laser sources (561 nm/488 nm) for stimulation and imaging into the proximal end of the fiber bundle while a set of galvanometer-scanning mirrors was used to couple the light stimulus to distinct fibers. A micro lens aided in focusing the light at the neurons. In vivo studies utilized C1V1(E122T/E162T)-TS-p2A-mCherry (Karl Deisseroth, Stanford) and GCaMP6s transgenic mice (Jackson Labs) for this all-optical approach. Our results demonstrate that imaging fiber bundles provide superior control of spatial selectivity of light delivery to specific neurons, and function as a conduit for optical imaging and recording at the in vivo site of stimulation, in contrast to the use of single multimode fibers that diffusely illuminate tissue and lack in vivo imaging capabilities.

Research paper thumbnail of Spatially controlled optogenetic light stimulation and recording platform via imaging fiber bundles (Conference Presentation)

Clinical and Translational Neurophotonics; Neural Imaging and Sensing; and Optogenetics and Optical Manipulation, 2016

Current methods for light delivery in in vivo optogenetic applications are typically accomplished... more Current methods for light delivery in in vivo optogenetic applications are typically accomplished via a single multimode fiber that diffuses light over a large area of the brain, and relies on the spatial distribution of transfected light-sensitive neurons for targeted control. In our investigations, an imaging fiber bundle (Schott) containing 4,500 individual fibers, each with a diameter of 7.5 µm, and an overall outer bundle diameter of 530 µm, served as a conduit for light delivery and optical recording/imaging. The use of this fiber bundle, in contrast to a single multimode fiber, allows for individually-addressable fibers, spatial selectivity at the stimulus site, more precise control of light delivery, and full field-of-view imaging and/or optical recordings of individual neurons in local neural circuits. An objective coupled the two continuous wave diode laser sources (561nm/488nm) (Coherent) for stimulation and imaging into the fiber bundle while a set of galvanometer-scanning mirrors was used to couple the light stimulus to distinct fibers within the proximal end of the imaging fiber bundle. In our study, C1V1(E122T/E162T)-TS-p2A-mCherry (Karl Deisseroth, Stanford) and GCaMP6s transgenic mice (Jackson Labs) were utilized for this all-optical approach. The results of our investigation demonstrate that imaging fiber bundles provide a new level of spatial selectivity and control of light delivery to specific neurons, as well as function as a conduit for optical imaging and recording at the in vivo site of stimulation, in contrast to the use of single multimode fibers that diffusely illuminate neural tissue and lack in vivo imaging capabilities.

Research paper thumbnail of Spatial and Temporal Analyses of Slow Electrical Oscillations Recorded from In-Vitro Neuronal Networks

Biophysical Journal, 2015

Research paper thumbnail of Dynamic Tracking Algorithm for Time-Varying Neuronal Network Connectivity using Wide-Field Optical Image Video Sequences

Scientific Reports, 2020

Propagation of signals between neurons and brain regions provides information about the functiona... more 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 comp...

Research paper thumbnail of Coherent control of an opsin in living brain tissue

Nature Physics, 2017

Retinal-based opsins are light-sensitive proteins. The photoisomerization reaction of these prote... more Retinal-based opsins are light-sensitive proteins. The photoisomerization reaction of these proteins has been studied outside cellular environments using ultrashort tailored light pulses 1-5. 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. Controlling cell functions with light has been achieved in recent years using natural and synthetic photosensitive proteins 6,7 , and retinal-based opsins, such as ChR2, are at the centre of this field. Usually mutant opsins are genetically engineered to improve cell activation at a specific wavelength or to make the protein predominantly absorb a different colour of light; parameters governing non

Research paper thumbnail of Fluctuation Correlation Spectroscopy with a Laser-Scanning Microscope: Exploiting the Hidden Time Structure

Biophysical Journal, 2005

Images obtained with a laser-scanning microscope contain a time structure that can be exploited t... more 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.

Research paper thumbnail of Measuring Fast Dynamics in Solutions and Cells with a Laser Scanning Microscope

Biophysical Journal, 2005

Single-point fluorescence correlation spectroscopy (FCS) allows measurements of fast diffusion an... more 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.

Research paper thumbnail of Regulation of G Protein Signals by Membrane Domains

Research paper thumbnail of 1 Biophysical Journal, Volume 96 Supporting Material EGFR Juxtamembrane Domain, Membranes and Calmodulin: Kinetics of Their Interaction

bound to phospholipid vesicles Ca2+/CaM rips the fluorescent peptide from the phospholipid vesicl... more bound to phospholipid vesicles Ca2+/CaM rips the fluorescent peptide from the phospholipid vesicles Mixing chamber FIGURE S1 Cartoon illustrating the stopped flow experiments designed to measure the effect of Ca/CaM on the rate of peptide dissociation from vesicles. Acrylodan-labeled EGFR(645-660) peptides bound to phospholipid vesicles are in one syringe and calcium/calmodulin is in the other syringe. The acrylodan fluorescence is quenched when the peptide is bound to the membrane, which contains NBD-PS. Upon mixing the solutions together rapidly, calcium/calmodulin rips the peptide from the surface and allows the fluorescence to increase, which is followed as a function of time. The dead time of the apparatus is ~ 5 ms. Peptide labeling and synthesis. To attach the acyrlodan label to EGFR(645-660), 1mg of peptide was dissolved in 0.8mL 10mM K2HPO4/KH2PO4, pH 7 buffer, mixed with the probe dissolved in DMF (1:1 probe:peptide molar ratio) and incubated for at least 1h. Labeled pepti...

Research paper thumbnail of Optogenetic skeletal muscle-powered adaptive biological machines

Proceedings of the National Academy of Sciences, 2016

Significance Understanding the design rules that govern the structure and function of natural bio... more Significance Understanding the design rules that govern the structure and function of natural biological systems gives us the ability to forward engineer machines integrated with and powered by biological components. Such machines, or “bio-bots,” can sense, process, and respond to dynamic environmental signals in real time, enabling a variety of applications. Here we present a modular optogenetic muscle actuator used to power actuation and locomotion of 3D printed flexible skeletons. Observing and controlling the functional response of such muscle-powered machines helps replicate the complex adaptive functionality we observe in natural biological systems. This demonstration thus sets the stage for building the next generation of bio-integrated machines and systems targeted at a diverse array of functional tasks.

Research paper thumbnail of Optogenetic Stimulation of Channelrhodopsin-2 Expressing Neurons Flips Cortical Networks from Low to High Activity State

Biophysical Journal, 2014

Research paper thumbnail of Role of caveolae in the duration of g protein signals

Research paper thumbnail of Electrical properties and the surface characteristics of lanthanum oxide/water interface

Journal of Colloid and Interface Science, 1988

Etude de l'adsorption des ions OH − et H + sur une electrode de verre recouverte d'oxyde ... more Etude de l'adsorption des ions OH − et H + sur une electrode de verre recouverte d'oxyde de lanthane (III), en fonction du pH et de la force ionique de la solution

Research paper thumbnail of Signaling through a G Protein-coupled Receptor and Its Corresponding G Protein Follows a Stoichiometrically Limited Model

Journal of Biological Chemistry, 2007

Research paper thumbnail of EGFR Juxtamembrane Domain, Membranes, and Calmodulin: Kinetics of Their Interaction

Biophysical Journal, 2009

Calcium/calmodulin (Ca/CaM) binds to the intracellular juxtamembrane domain (JMD) of the epiderma... more 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, [Ca 2þ ] 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.

Research paper thumbnail of The ATP-cytokine-adenosine hypothesis: How the brain translates past activity into sleep

Sleep and Biological Rhythms, 2011

Tumor necrosis factor alpha (TNF), interleukin-1 beta (IL1), and other cytokines are involved in ... more Tumor necrosis factor alpha (TNF), interleukin-1 beta (IL1), and other cytokines are involved in non-rapid eye movement sleep (NREM) regulation under physiological and inflammatory conditions. Brain levels of IL1 and TNF increase with prolonged wakefulness. Injection of exogenous IL1 or TNF, mimicking sleep loss, induces sleepiness, excess sleep, fatigue, poor cognition, and enhanced sensitivity to pain. These symptoms characterize the syndrome associated with sleep loss. Extracellular ATP released during neuro-and glio-transmission, acting via purine P2 receptors on glia, releases IL1 and TNF. This extracellular ATP mechanism may provide an index of activity used by the brain to keep track of prior wakefulness. Prolonged wakefulness is associated with enhanced neuronal activity. TNF and IL1, in turn, act on neurons to change their intrinsic properties and sensitivities to neurotransmitters and neuromodulators such as adenosine and glutamate. Such actions change network input-output properties (i.e. state shift). State oscillations, for instance, occur within cortical columns and are responsive to TNF. Sleep is thus viewed as a local usedependent process regulated in part by cytokines. Further, state oscillations are viewed as a fundamental process of any neuronal/glia network. To investigate these hypotheses we developed an in vitro neuronal/glia culture system exhibiting field potential oscillations and have mathematically modeled the local use-dependent view of sleep initiation. These views have profound implications for sleep pathologies and function.

Research paper thumbnail of Optogenetic Stimulation Enhances Interleukin-1beta Levels in Cultured Neurons

Biophysical Journal, 2011

recordings now reliably deliver simultaneous signals from a hundred or more neurons or networks. ... more recordings now reliably deliver simultaneous signals from a hundred or more neurons or networks. However, many analytic techniques are presently computationally limited to smaller numbers of signals, severely limiting our ability to relate these neural signals to brain functions including sensation, perception, decision, and action. To address this imbalance, we are developing a new open source Neurophysiology Extended Analysis Tool. NEAT leverages existing code bases and new massively-parallel computational technology to enable any multielectrode lab to perform high-throughput informative analyses. We begin with linked evaluations of: 1) computational bottlenecks in analytic routines, many information-theoretic, developed for our Spike Train Analysis Toolkit and distributed via neuroanalysis.org to over 1,600 sites [Goldberg et al. Neuroinformatics 7, 165-178, 2009], and 2) specific capabilities and restrictions of new graphics-processor-derived computational engines supplied on inexpensive drop-in cards. We project a greater than order of magnitude speedup that will allow many offline analyses to be performed in real time during experiments, and now-impractical questions to be explored offline in reasonable compute times. For example, pairwise analyses now possible on 10 or fewer neurons may be extendible to 50 or more. We focus on information-theoretic measures and standard pairwise correlations, JPSTHs, spectra, coherences, and new and significant analyses that present significant loads for multineuron recordings. To aid communities planning similar GPU-enabled analyses, we note that the complex, structured, and hierarchic GPU architecture requires special optimization strategies: decomposing code into >1,000 simultaneous threads is needed to efficiently use the 448 cores on new GPUs, data should be loaded into on-chip memory once and re-used, avoiding transfers to other memory layers, kernel processes must optimize thread/kernel and thread/block instruction execution in few clock cycles, flow control code should control multi-thread warps, not individual threads. Support: MH057153/MH068012.

Research paper thumbnail of Computational algorithm for assessing inter-neuronal connectivity to optimize optogenetic stimulation and neural circuit activity (Conference Presentation)

Optogenetics and Optical Manipulation 2018, 2018

All-optical systems for stimulating and imaging neuronal activity have served as powerful tools f... more All-optical systems for stimulating and imaging neuronal activity have served as powerful tools for understanding the underlying circuitry of the brain. Experiments using these setups, however, tend to choose stimulation locations based solely on what brain regions are of interest, and take for granted that stimulation effects may vary even within localized brain regions. We thus have developed an algorithm for acquiring neuronal activity via calcium imaging data to assess network connectivity. These parameters include the signal rise time, decay time, inter-event intervals, and the timing and amplitude of signal peaks. These parameters are then compared between cell clusters for similarities, and used as a basis for establishing interconnectivity. Additionally, we have incorporated both temporal and spatial correlation functions to assess inter-neuronal connectivity based on these parameters. This data is then run through a genetic algorithm, applying weights to cells with similar parameters to learn which are interconnected in a given field-of-view. For this study, hippocampal neurons extracted from 2 day old transgenic mice (GCaMP6s, Jackson Labs), - cultured for 2 weeks and imaged under single and two-photon conditions. Single-photon imaging was performed under a commercial Zeiss microscope, whereas two-photon imaging was performed with an in-house imaging system. Results demonstrate a strong correlation between these parameters and cellular connectivity, making them noteworthy markers for targeted stimulation. This study demonstrates an efficient method of assessing network connectivity for various imaging techniques, and hence directed targeting for optogenetic stimulation.

Research paper thumbnail of Reconstruction and depixelation of multicore fiber bundle images by fiber bundle rotation (Conference Presentation)

Multicore fiber bundles for imaging and stimulating optogenetically modified neurons have been la... more Multicore fiber bundles for imaging and stimulating optogenetically modified neurons have been largely adopted in neurophotonics research. They allow for directed, single-cell stimulation and imaging of neuronal activity. An inherent limitation of these bundles is the presence and detection of the empty space between individual fibers, resulting in a loss of significant amounts of data, and reduced image quality due to pixilation effects. We propose a novel approach and algorithm to depixelation and image reconstruction from fiber bundles that utilizes multiple image frames collected during on-axis fiber bundle rotation. The approach involves first acquiring the Fourier transform of a stationary, unrotated image, followed by its rotated counterparts. The phase information from each image is then acquired, cross-correlated, and the angle of rotation determined from this correlation. Rotated images are then weighed and summed to generate a final reconstructed, depixelated image. Simul...

Research paper thumbnail of Optical excitation and detection of neuronal activity

Journal of biophotonics, Jan 12, 2018

Optogenetics has emerged as an exciting tool for manipulating neural activity, which in turn, can... more 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 changes with nanometer scale sensitivity. We found that optical pathlength 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...

Research paper thumbnail of Spatial control of in vivo optogenetic light stimulation and recording via an imaging fiber bundle (Conference Presentation)

Optogenetics and Optical Manipulation, 2017

Light delivery in in vivo optogenetic applications are typically accomplished via a single multim... more Light delivery in in vivo optogenetic applications are typically accomplished via a single multimode fiber that diffuses light over a large area of the brain, and relies heavily on the spatial distribution of transfected light-sensitive neurons for targeted control. In our investigations, an imaging fiber bundle (Schott, 1534702) containing 4,500 individual fibers, each with a diameter of 7.5 µm, and an overall outer bundle diameter of 530 µm, was used as the conduit for light delivery and optical recording/imaging in neuron cultures and in in vivo mouse brain. We demonstrated that the use of this fiber bundle, in contrast to a single multimode fiber, allowed for individually-addressable fibers, spatial selectivity at the stimulus site, precise control of light delivery, and full field-of-view imaging and/or optical recordings of neurons. An objective coupled the two continuous wave diode laser sources (561 nm/488 nm) for stimulation and imaging into the proximal end of the fiber bundle while a set of galvanometer-scanning mirrors was used to couple the light stimulus to distinct fibers. A micro lens aided in focusing the light at the neurons. In vivo studies utilized C1V1(E122T/E162T)-TS-p2A-mCherry (Karl Deisseroth, Stanford) and GCaMP6s transgenic mice (Jackson Labs) for this all-optical approach. Our results demonstrate that imaging fiber bundles provide superior control of spatial selectivity of light delivery to specific neurons, and function as a conduit for optical imaging and recording at the in vivo site of stimulation, in contrast to the use of single multimode fibers that diffusely illuminate tissue and lack in vivo imaging capabilities.

Research paper thumbnail of Spatially controlled optogenetic light stimulation and recording platform via imaging fiber bundles (Conference Presentation)

Clinical and Translational Neurophotonics; Neural Imaging and Sensing; and Optogenetics and Optical Manipulation, 2016

Current methods for light delivery in in vivo optogenetic applications are typically accomplished... more Current methods for light delivery in in vivo optogenetic applications are typically accomplished via a single multimode fiber that diffuses light over a large area of the brain, and relies on the spatial distribution of transfected light-sensitive neurons for targeted control. In our investigations, an imaging fiber bundle (Schott) containing 4,500 individual fibers, each with a diameter of 7.5 µm, and an overall outer bundle diameter of 530 µm, served as a conduit for light delivery and optical recording/imaging. The use of this fiber bundle, in contrast to a single multimode fiber, allows for individually-addressable fibers, spatial selectivity at the stimulus site, more precise control of light delivery, and full field-of-view imaging and/or optical recordings of individual neurons in local neural circuits. An objective coupled the two continuous wave diode laser sources (561nm/488nm) (Coherent) for stimulation and imaging into the fiber bundle while a set of galvanometer-scanning mirrors was used to couple the light stimulus to distinct fibers within the proximal end of the imaging fiber bundle. In our study, C1V1(E122T/E162T)-TS-p2A-mCherry (Karl Deisseroth, Stanford) and GCaMP6s transgenic mice (Jackson Labs) were utilized for this all-optical approach. The results of our investigation demonstrate that imaging fiber bundles provide a new level of spatial selectivity and control of light delivery to specific neurons, as well as function as a conduit for optical imaging and recording at the in vivo site of stimulation, in contrast to the use of single multimode fibers that diffusely illuminate neural tissue and lack in vivo imaging capabilities.

Research paper thumbnail of Spatial and Temporal Analyses of Slow Electrical Oscillations Recorded from In-Vitro Neuronal Networks

Biophysical Journal, 2015

Research paper thumbnail of Dynamic Tracking Algorithm for Time-Varying Neuronal Network Connectivity using Wide-Field Optical Image Video Sequences

Scientific Reports, 2020

Propagation of signals between neurons and brain regions provides information about the functiona... more 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 comp...

Research paper thumbnail of Coherent control of an opsin in living brain tissue

Nature Physics, 2017

Retinal-based opsins are light-sensitive proteins. The photoisomerization reaction of these prote... more Retinal-based opsins are light-sensitive proteins. The photoisomerization reaction of these proteins has been studied outside cellular environments using ultrashort tailored light pulses 1-5. 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. Controlling cell functions with light has been achieved in recent years using natural and synthetic photosensitive proteins 6,7 , and retinal-based opsins, such as ChR2, are at the centre of this field. Usually mutant opsins are genetically engineered to improve cell activation at a specific wavelength or to make the protein predominantly absorb a different colour of light; parameters governing non

Research paper thumbnail of Fluctuation Correlation Spectroscopy with a Laser-Scanning Microscope: Exploiting the Hidden Time Structure

Biophysical Journal, 2005

Images obtained with a laser-scanning microscope contain a time structure that can be exploited t... more 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.

Research paper thumbnail of Measuring Fast Dynamics in Solutions and Cells with a Laser Scanning Microscope

Biophysical Journal, 2005

Single-point fluorescence correlation spectroscopy (FCS) allows measurements of fast diffusion an... more 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.