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1.  High fidelity optogenetic control of individual prefrontal cortical pyramidal neurons in vivo  
F1000Research  2012;1:7.
Precise spatial and temporal manipulation of neural activity in specific genetically defined cell populations is now possible with the advent of optogenetics. The emerging field of optogenetics consists of a set of naturally-occurring and engineered light-sensitive membrane proteins that are able to activate (e.g. channelrhodopsin-2, ChR2) or silence (e.g. halorhodopsin, NpHR) neural activity. Here we demonstrate the technique and the feasibility of using novel adeno-associated viral (AAV) tools to activate (AAV-CaMKllα-ChR2-eYFP) or silence (AAV-CaMKllα-eNpHR3.0-eYFP) neural activity of rat prefrontal cortical prelimbic (PL) pyramidal neurons  in vivo.  In vivo single unit extracellular recording of ChR2-transduced pyramidal neurons showed that delivery of brief (10 ms) blue (473 nm) light-pulse trains up to 20 Hz via a custom fiber optic-coupled recording electrode (optrode) induced spiking with high fidelity at 20 Hz for the duration of recording (up to two hours in some cases). To silence spontaneously active neurons, we transduced them with the NpHR construct and administered continuous green (532 nm) light to completely inhibit action potential activity for up to 10 seconds with 100% fidelity in most cases. These versatile photosensitive tools, combined with optrode recording methods, provide experimental control over activity of genetically defined neurons and can be used to investigate the functional relationship between neural activity and complex cognitive behavior.
doi:10.12688/f1000research.1-7.v1
PMCID: PMC3894804  PMID: 24555016
2.  A 3D glass optrode array for optical neural stimulation 
Biomedical Optics Express  2012;3(12):3087-3104.
This paper presents optical characterization of a first-generation SiO2 optrode array as a set of penetrating waveguides for both optogenetic and infrared (IR) neural stimulation. Fused silica and quartz discs of 3-mm thickness and 50-mm diameter were micromachined to yield 10 × 10 arrays of up to 2-mm long optrodes at a 400-μm pitch; array size, length and spacing may be varied along with the width and tip angle. Light delivery and loss mechanisms through these glass optrodes were characterized. Light in-coupling techniques include using optical fibers and collimated beams. Losses involve Fresnel reflection, coupling, scattering and total internal reflection in the tips. Transmission efficiency was constant in the visible and near-IR range, with the highest value measured as 71% using a 50-μm multi-mode in-coupling fiber butt-coupled to the backplane of the device. Transmittance and output beam profiles of optrodes with different geometries was investigated. Length and tip angle do not affect the amount of output power, but optrode width and tip angle influence the beam size and divergence independently. Finally, array insertion in tissue was performed to demonstrate its robustness for optical access in deep tissue.
doi:10.1364/BOE.3.003087
PMCID: PMC3521295  PMID: 23243561
(170.3890) Medical optics instrumentation; (220.4610) Optical fabrication; (230.7380) Waveguides, channeled; (170.3660) Light propagation in tissues
3.  Characterization of a 3D optrode array for infrared neural stimulation 
Biomedical Optics Express  2012;3(9):2200-2219.
This paper characterizes the Utah Slant Optrode Array (USOA) as a means to deliver infrared light deep into tissue. An undoped crystalline silicon (100) substrate was used to fabricate 10 × 10 arrays of optrodes with rows of varying lengths from 0.5 mm to 1.5 mm on a 400-μm pitch. Light delivery from optical fibers and loss mechanisms through these Si optrodes were characterized, with the primary loss mechanisms being Fresnel reflection, coupling, radiation losses from the tapered shank and total internal reflection in the tips. Transmission at the optrode tips with different optical fiber core diameters and light in-coupling interfaces was investigated. At λ = 1.55μm, the highest optrode transmittance of 34.7%, relative to the optical fiber output power, was obtained with a 50-μm multi-mode fiber butt-coupled to the optrode through an intervening medium of index n = 1.66. Maximum power is directed into the optrodes when using fibers with core diameters of 200 μm or less. In addition, the output power varied with the optrode length/taper such that longer and less tapered optrodes exhibited higher light transmission efficiency. Output beam profiles and potential impacts on physiological tests were also examined. Future work is expected to improve USOA efficiency to greater than 64%.
doi:10.1364/BOE.3.002200
PMCID: PMC3447562  PMID: 23024914
(170.3890) Medical optics instrumentation; (220.4610) Optical fabrication; (230.7380) Waveguides, channeled; (260.3060) Infrared
4.  Optical Measurements of Intramural Action Potentials in Isolated Porcine Hearts Using Optrodes 
Background
Measurements of intramural Vm would greatly increase knowledge of cardiac arrhythmias and defibrillation. Optrodes offer the possibility for three-dimensional Vm mapping but their signal quality has been inadequate.
Objectives
The objectives of this work were to improve optrode signal quality and use optrodes to measure intramural distribution of action potentials and shock-induced Vm changes in porcine hearts.
Methods
Optrodes were made from seven optical fibers 225 or 325 μm in diameter. Fiber ends were polished at 45° angle which improved light collection and allowed their insertion without a needle. Fluorescent measurements were performed in isolated porcine hearts perfused with Tyrode’s solution or blood using Vm-sensitive dye RH-237 and a 200-W Hg/Xe lamp.
Results
The signal-to-noise ratio for 325-μm fibers was 44±23 in blood-perfused hearts (n=5) and 106±45 in Tyrode-perfused hearts (n=3), which represents a ≈4-fold improvement over previously reported data. There was close correspondence between optical and electrical measurements of activation times and action potential duration (APD). No significant intramural APD gradients were observed at cycle lengths up to 4 s and in the presence of dofetilide or d-sotalol. Application of shocks (5–50 V/cm) produced large intramural Vm changes (up to ≈200%APA) possibly reflecting a combined effect of tissue discontinuities and optrode geometry.
Conclusions
A substantial improvement of optrode signal quality was achieved. Optical measurements of APD and activation times matched electrical measurements. Optrode measurements revealed no significant intramural APD gradients. Application of shocks caused large intramural Vm changes that could be influenced by the optrode geometry.
doi:10.1016/j.hrthm.2007.07.002
PMCID: PMC2098877  PMID: 17954403
cardiac excitation; action potentials; defibrillation; virtual electrodes; optical mapping; fiber optics
5.  Physical principles for scalable neural recording 
Simultaneously measuring the activities of all neurons in a mammalian brain at millisecond resolution is a challenge beyond the limits of existing techniques in neuroscience. Entirely new approaches may be required, motivating an analysis of the fundamental physical constraints on the problem. We outline the physical principles governing brain activity mapping using optical, electrical, magnetic resonance, and molecular modalities of neural recording. Focusing on the mouse brain, we analyze the scalability of each method, concentrating on the limitations imposed by spatiotemporal resolution, energy dissipation, and volume displacement. Based on this analysis, all existing approaches require orders of magnitude improvement in key parameters. Electrical recording is limited by the low multiplexing capacity of electrodes and their lack of intrinsic spatial resolution, optical methods are constrained by the scattering of visible light in brain tissue, magnetic resonance is hindered by the diffusion and relaxation timescales of water protons, and the implementation of molecular recording is complicated by the stochastic kinetics of enzymes. Understanding the physical limits of brain activity mapping may provide insight into opportunities for novel solutions. For example, unconventional methods for delivering electrodes may enable unprecedented numbers of recording sites, embedded optical devices could allow optical detectors to be placed within a few scattering lengths of the measured neurons, and new classes of molecularly engineered sensors might obviate cumbersome hardware architectures. We also study the physics of powering and communicating with microscale devices embedded in brain tissue and find that, while radio-frequency electromagnetic data transmission suffers from a severe power–bandwidth tradeoff, communication via infrared light or ultrasound may allow high data rates due to the possibility of spatial multiplexing. The use of embedded local recording and wireless data transmission would only be viable, however, given major improvements to the power efficiency of microelectronic devices.
doi:10.3389/fncom.2013.00137
PMCID: PMC3807567  PMID: 24187539
neural recording; brain activity mapping; electrical recording; optical methods; magnetic resonance imaging; molecular recording; embedded electronics
6.  In Vivo Optogenetic Control of Striatal and Thalamic Neurons in Non-Human Primates 
PLoS ONE  2012;7(11):e50808.
Electrical and pharmacological stimulation methods are commonly used to study neuronal brain circuits in vivo, but are problematic, because electrical stimulation has limited specificity, while pharmacological activation has low temporal resolution. A recently developed alternative to these methods is the use of optogenetic techniques, based on the expression of light sensitive channel proteins in neurons. While optogenetics have been applied in in vitro preparations and in in vivo studies in rodents, their use to study brain function in nonhuman primates has been limited to the cerebral cortex. Here, we characterize the effects of channelrhodopsin-2 (ChR2) transfection in subcortical areas, i.e., the putamen, the external globus pallidus (GPe) and the ventrolateral thalamus (VL) of rhesus monkeys. Lentiviral vectors containing the ChR2 sequence under control of the elongation factor 1α promoter (pLenti-EF1α -hChR2(H134R)-eYFP-WPRE, titer 109 particles/ml) were deposited in GPe, putamen and VL. Four weeks later, a probe combining a conventional electrode and an optic fiber was introduced in the previously injected brain areas. We found light-evoked responses in 31.5% and 32.7% of all recorded neurons in the striatum and thalamus, respectively, but only in 2.5% of recorded GPe neurons. As expected, most responses were time-locked increases in firing, but decreases or mixed responses were also seen, presumably via ChR2-mediated activation of local inhibitory connections. Light and electron microscopic analyses revealed robust expression of ChR2 on the plasma membrane of cell somas, dendrites, spines and terminals in the striatum and VL. This study demonstrates that optogenetic experiments targeting the striatum and basal ganglia-related thalamic nuclei can be successfully achieved in monkeys. Our results indicate important differences of the type and magnitude of responses in each structure. Experimental conditions such as the vector used, the number and rate of injections, or the light stimulation conditions have to be optimized for each structure studied.
doi:10.1371/journal.pone.0050808
PMCID: PMC3511281  PMID: 23226390
7.  Computational Optogenetics: Empirically-Derived Voltage- and Light-Sensitive Channelrhodopsin-2 Model 
PLoS Computational Biology  2013;9(9):e1003220.
Channelrhodospin-2 (ChR2), a light-sensitive ion channel, and its variants have emerged as new excitatory optogenetic tools not only in neuroscience, but also in other areas, including cardiac electrophysiology. An accurate quantitative model of ChR2 is necessary for in silico prediction of the response to optical stimulation in realistic tissue/organ settings. Such a model can guide the rational design of new ion channel functionality tailored to different cell types/tissues. Focusing on one of the most widely used ChR2 mutants (H134R) with enhanced current, we collected a comprehensive experimental data set of the response of this ion channel to different irradiances and voltages, and used these data to develop a model of ChR2 with empirically-derived voltage- and irradiance- dependence, where parameters were fine-tuned via simulated annealing optimization. This ChR2 model offers: 1) accurate inward rectification in the current-voltage response across irradiances; 2) empirically-derived voltage- and light-dependent kinetics (activation, deactivation and recovery from inactivation); and 3) accurate amplitude and morphology of the response across voltage and irradiance settings. Temperature-scaling factors (Q10) were derived and model kinetics was adjusted to physiological temperatures. Using optical action potential clamp, we experimentally validated model-predicted ChR2 behavior in guinea pig ventricular myocytes. The model was then incorporated in a variety of cardiac myocytes, including human ventricular, atrial and Purkinje cell models. We demonstrate the ability of ChR2 to trigger action potentials in human cardiomyocytes at relatively low light levels, as well as the differential response of these cells to light, with the Purkinje cells being most easily excitable and ventricular cells requiring the highest irradiance at all pulse durations. This new experimentally-validated ChR2 model will facilitate virtual experimentation in neural and cardiac optogenetics at the cell and organ level and provide guidance for the development of in vivo tools.
Author Summary
Optogenetics, the use of light-sensitive ion channels for stimulation of mammalian cells and tissues, offers specificity and superior precision of control compared to traditional chemical or electrical means of stimulation. In particular, Channelrhodospin-2 (ChR2), a light-sensitive ion channel, originally derived from algae, has found wide-spread application in neuroscience for controlled stimulation of different brain regions. More recently, this work was extended to other organs, including the heart, where it opens the possibility for a new generation of optical pacemakers. The development of new optogenetic tools that allow for more efficient optical stimulation can be guided by computational prediction of the response of different cells and tissues to light. In this report, we provide a new computational model of ChR2 that was empirically validated and can be inserted into different cell types – neurons or heart cells – for virtual optical stimulation and prediction of optimal light-delivery arrangements, minimum energy needs etc. Overall, virtual optogenetics can accelerate the development of new optical stimulation tools for better understanding and control of brain and heart function.
doi:10.1371/journal.pcbi.1003220
PMCID: PMC3772068  PMID: 24068903
8.  Saccade Modulation by Optical and Electrical Stimulation in the Macaque Frontal Eye Field 
The Journal of Neuroscience  2013;33(42):16684-16697.
Recent studies have demonstrated that strong neural modulations can be evoked with optogenetic stimulation in macaque motor cortex without observing any evoked movements (Han et al., 2009, 2011; Diester et al., 2011). It remains unclear why such perturbations do not generate movements and if conditions exist under which they may evoke movements. In this study, we examine the effects of five optogenetic constructs in the macaque frontal eye field and use electrical microstimulation to assess whether optical perturbation of the local network leads to observable motor changes during optical, electrical, and combined stimulation. We report a significant increase in the probability of evoking saccadic eye movements when low current electrical stimulation is coupled to optical stimulation compared with when electrical stimulation is used alone. Experiments combining channelrhodopsin 2 (ChR2) and electrical stimulation with simultaneous fMRI revealed no discernible fMRI activity at the electrode tip with optical stimulation but strong activity with electrical stimulation. Our findings suggest that stimulation with current ChR2 optogenetic constructs generates subthreshold activity that contributes to the initiation of movements but, in most cases, is not sufficient to evoke a motor response.
doi:10.1523/JNEUROSCI.2675-13.2013
PMCID: PMC3797379  PMID: 24133271
9.  A Microfluidic Microelectrode Array for Simultaneous Electrophysiology, Chemical Stimulation, and Imaging of Brain Slices 
Lab on a chip  2013;13(4):527-535.
In order to understand information processing in neural circuits, it is necessary to detect both electrical and chemical signaling with high spatial and temporal resolution. Although the primary currency of neural information processing is electrical, many of the downstream effects of the electrical signals on the circuits that generate them are dependent on activity-dependent increases in intracellular calcium concentration. It is therefore of great utility to be able to record electrical signals in neural circuits at multiple sites while at the same time detecting optical signals from reporters of intracellular calcium levels. We describe here a microfluidic multi-electrode array (MMEA) capable of high-resolution extracellular recording from brain slices that is optically compatible with calcium imaging at single cell resolution. We show the application of the MMEA device to record waves of spontaneous activity in developing cortical slices and to perform multi-site extracellular recordings during simultaneous calcium imaging of activity. The MMEA has the unique capability to simultaneously allow focal electrical and chemical stimuli at different locations of the surface of a brain slice.
doi:10.1039/c2lc40826k
PMCID: PMC3737778  PMID: 23042571
10.  Closed-Loop, Open-Source Electrophysiology 
Multiple extracellular microelectrodes (multi-electrode arrays, or MEAs) effectively record rapidly varying neural signals, and can also be used for electrical stimulation. Multi-electrode recording can serve as artificial output (efferents) from a neural system, while complex spatially and temporally targeted stimulation can serve as artificial input (afferents) to the neuronal network. Multi-unit or local field potential (LFP) recordings can not only be used to control real world artifacts, such as prostheses, computers or robots, but can also trigger or alter subsequent stimulation. Real-time feedback stimulation may serve to modulate or normalize aberrant neural activity, to induce plasticity, or to serve as artificial sensory input. Despite promising closed-loop applications, commercial electrophysiology systems do not yet take advantage of the bidirectional capabilities of multi-electrodes, especially for use in freely moving animals. We addressed this lack of tools for closing the loop with NeuroRighter, an open-source system including recording hardware, stimulation hardware, and control software with a graphical user interface. The integrated system is capable of multi-electrode recording and simultaneous patterned microstimulation (triggered by recordings) with minimal stimulation artifact. The potential applications of closed-loop systems as research tools and clinical treatments are broad; we provide one example where epileptic activity recorded by a multi-electrode probe is used to trigger targeted stimulation, via that probe, to freely moving rodents.
doi:10.3389/fnins.2010.00031
PMCID: PMC2940414  PMID: 20859448
multi-electrode array; stimulation; epilepsy; closed-loop; artifact
11.  Multiple-Color Optical Activation, Silencing, and Desynchronization of Neural Activity, with Single-Spike Temporal Resolution 
PLoS ONE  2007;2(3):e299.
The quest to determine how precise neural activity patterns mediate computation, behavior, and pathology would be greatly aided by a set of tools for reliably activating and inactivating genetically targeted neurons, in a temporally precise and rapidly reversible fashion. Having earlier adapted a light-activated cation channel, channelrhodopsin-2 (ChR2), for allowing neurons to be stimulated by blue light, we searched for a complementary tool that would enable optical neuronal inhibition, driven by light of a second color. Here we report that targeting the codon-optimized form of the light-driven chloride pump halorhodopsin from the archaebacterium Natronomas pharaonis (hereafter abbreviated Halo) to genetically-specified neurons enables them to be silenced reliably, and reversibly, by millisecond-timescale pulses of yellow light. We show that trains of yellow and blue light pulses can drive high-fidelity sequences of hyperpolarizations and depolarizations in neurons simultaneously expressing yellow light-driven Halo and blue light-driven ChR2, allowing for the first time manipulations of neural synchrony without perturbation of other parameters such as spiking rates. The Halo/ChR2 system thus constitutes a powerful toolbox for multichannel photoinhibition and photostimulation of virally or transgenically targeted neural circuits without need for exogenous chemicals, enabling systematic analysis and engineering of the brain, and quantitative bioengineering of excitable cells.
doi:10.1371/journal.pone.0000299
PMCID: PMC1808431  PMID: 17375185
12.  A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo 
Optogenetic methods have emerged as a powerful tool for elucidating neural circuit activity underlying a diverse set of behaviors across a broad range of species. Optogenetic tools of microbial origin consist of light-sensitive membrane proteins that are able to activate (e.g., channelrhodopsin-2, ChR2) or silence (e.g., halorhodopsin, NpHR) neural activity ingenetically-defined cell types over behaviorally-relevant timescales. We first demonstrate a simple approach for adeno-associated virus-mediated delivery of ChR2 and NpHR transgenes to the dorsal subiculum and prelimbic region of the prefrontal cortex in rat. Because ChR2 and NpHR are genetically targetable, we describe the use of this technology to control the electrical activity of specific populations of neurons (i.e., pyramidal neurons) embedded in heterogeneous tissue with high temporal precision. We describe herein the hardware, custom software user interface, and procedures that allow for simultaneous light delivery and electrical recording from transduced pyramidal neurons in an anesthetized in vivo preparation. These light-responsive tools provide the opportunity for identifying the causal contributions of different cell types to information processing and behavior.
doi:10.3791/50291
PMCID: PMC3857391  PMID: 24022017
Neuroscience; Issue 79; Genetic Techniques; Genetics; Behavioral; Biological Science Disciplines; Neurosciences; genetics (animal and plant); Investigative Techniques; Behavior and Behavior Mechanisms; Behavioral Disciplines and Activities; Natural Science Disciplines; Optogenetics; prefrontal cortex; subiculum; virus injection;  in vivo recording; Neurophysiology; prelimbic; optrode; molecular neurogenetics; Gene targeting
13.  Optogenetic stimulation of the auditory pathway 
The Journal of Clinical Investigation  2014;124(3):1114-1129.
Auditory prostheses can partially restore speech comprehension when hearing fails. Sound coding with current prostheses is based on electrical stimulation of auditory neurons and has limited frequency resolution due to broad current spread within the cochlea. In contrast, optical stimulation can be spatially confined, which may improve frequency resolution. Here, we used animal models to characterize optogenetic stimulation, which is the optical stimulation of neurons genetically engineered to express the light-gated ion channel channelrhodopsin-2 (ChR2). Optogenetic stimulation of spiral ganglion neurons (SGNs) activated the auditory pathway, as demonstrated by recordings of single neuron and neuronal population responses. Furthermore, optogenetic stimulation of SGNs restored auditory activity in deaf mice. Approximation of the spatial spread of cochlear excitation by recording local field potentials (LFPs) in the inferior colliculus in response to suprathreshold optical, acoustic, and electrical stimuli indicated that optogenetic stimulation achieves better frequency resolution than monopolar electrical stimulation. Virus-mediated expression of a ChR2 variant with greater light sensitivity in SGNs reduced the amount of light required for responses and allowed neuronal spiking following stimulation up to 60 Hz. Our study demonstrates a strategy for optogenetic stimulation of the auditory pathway in rodents and lays the groundwork for future applications of cochlear optogenetics in auditory research and prosthetics.
doi:10.1172/JCI69050
PMCID: PMC3934189  PMID: 24509078
14.  Optogenetic identification of striatal projection neuron subtypes during in vivo recordings 
Brain research  2012;1511:21-32.
Optogenetics has revolutionized neuroscience over the past several years by allowing researchers to modulate the activity of specific cell types, both in vitro and in vivo. One promising application of optogenetics is to use channelrhodopsin-2 (ChR2) mediated spiking to identify distinct cell types in electrophysiological recordings from awake behaving animals. In this paper, we apply this approach to in vivo recordings of the two major projection cell types in the striatum: the direct- and indirect-pathway medium spiny neurons. We expressed ChR2 in the neurons of the direct or indirect pathways using a cre-dependent viral strategy and performed electrical recordings together with optical stimulation using an implanted microwire array that included an integrated optical fiber. Despite the apparent simplicity of identifying ChR2-expressing neurons as those that respond to light, we encountered multiple potential confounds when applying this approach. Here, we describe and address these confounds and provide a Matlab tool so others can implement our analysis methods.
doi:10.1016/j.brainres.2012.11.018
PMCID: PMC3594574  PMID: 23178332
optogenetics; ChR2; striatum; in vivo recording
15.  Optogenetically Induced Seizure and the Longitudinal Hippocampal Network Dynamics 
PLoS ONE  2013;8(4):e60928.
Epileptic seizure is a paroxysmal and self-limited phenomenon characterized by abnormal hypersynchrony of a large population of neurons. However, our current understanding of seizure dynamics is still limited. Here we propose a novel in vivo model of seizure-like afterdischarges using optogenetics, and report on investigation of directional network dynamics during seizure along the septo-temporal (ST) axis of hippocampus. Repetitive pulse photostimulation was applied to the rodent hippocampus, in which channelrhodopsin-2 (ChR2) was expressed, under simultaneous recording of local field potentials (LFPs). Seizure-like afterdischarges were successfully induced after the stimulation in both W-TChR2V4 transgenic (ChR2V-TG) rats and in wild type rats transfected with adeno-associated virus (AAV) vectors carrying ChR2. Pulse frequency at 10 and 20 Hz, and a 0.05 duty ratio were optimal for afterdischarge induction. Immunohistochemical c-Fos staining after a single induced afterdischarge confirmed neuronal activation of the entire hippocampus. LFPs were recorded during seizure-like afterdischarges with a multi-contact array electrode inserted along the ST axis of hippocampus. Granger causality analysis of the LFPs showed a bidirectional but asymmetric increase in signal flow along the ST direction. State space presentation of the causality and coherence revealed three discrete states of the seizure-like afterdischarge phenomenon: 1) resting state; 2) afterdischarge initiation with moderate coherence and dominant septal-to-temporal causality; and 3) afterdischarge termination with increased coherence and dominant temporal-to-septal causality. A novel in vivo model of seizure-like afterdischarge was developed using optogenetics, which was advantageous in its reproducibility and artifact-free electrophysiological observations. Our results provide additional evidence for the potential role of hippocampal septo-temporal interactions in seizure dynamics in vivo. Bidirectional networks work hierarchically along the ST hippocampus in the genesis and termination of epileptic seizures.
doi:10.1371/journal.pone.0060928
PMCID: PMC3622611  PMID: 23593349
16.  Cortical Neurovascular Coupling Driven by Stimulation of Channelrhodopsin-2 
PLoS ONE  2012;7(9):e46607.
While functional imaging is widely used in studies of the brain, how well the hemodynamic signal represents the underlying neural activity is still unclear. And there is a debate on whether hemodynamic signal is more tightly related to synaptic activity or action potentials. This study intends to address these questions by examining neurovascular coupling driven by pyramidal cells in the motor cortex of rats. Pyramidal cells in the motor cortex of rats were selectively transduced with the light sensitive cation channel channelrhodopsin-2 (ChR2). Electrophysiological recordings and optical intrinsic signal imaging were performed simultaneously and synchronously to capture the neural activity and hemodynamics induced by optical stimulation of ChR2-expressing pyramidal cells. Our results indicate that both synaptic activity (local field potential, LFP) and action potentials (multi-unit activity, MUA) are tightly related to hemodynamic signals. While LFPs in γ band are better in predicting hemodynamic signals elicited by short stimuli, MUA has better predictions to hemodynamic signals elicited by long stimuli. Our results also indicate that strong nonlinearity exists in neurovascular coupling.
doi:10.1371/journal.pone.0046607
PMCID: PMC3460925  PMID: 23029556
17.  Long-term optical stimulation of channelrhodopsin-expressing neurons to study network plasticity 
Neuronal plasticity produces changes in excitability, synaptic transmission, and network architecture in response to external stimuli. Network adaptation to environmental conditions takes place in time scales ranging from few seconds to days, and modulates the entire network dynamics. To study the network response to defined long-term experimental protocols, we setup a system that combines optical and electrophysiological tools embedded in a cell incubator. Primary hippocampal neurons transduced with lentiviruses expressing channelrhodopsin-2/H134R were subjected to various photostimulation protocols in a time window in the order of days. To monitor the effects of light-induced gating of network activity, stimulated transduced neurons were simultaneously recorded using multi-electrode arrays (MEAs). The developed experimental model allows discerning short-term, long-lasting, and adaptive plasticity responses of the same neuronal network to distinct stimulation frequencies applied over different temporal windows.
doi:10.3389/fnmol.2013.00022
PMCID: PMC3747358  PMID: 23970852
long-term recordings; primary neurons; optogenetics; network activity; network plasticity
18.  Multi-array silicon probes with integrated optical fibers: light-assisted perturbation and recording of local neural circuits in the behaving animal 
The European journal of neuroscience  2010;31(12):2279-2291.
Recordings of large neuronal ensembles and neural stimulation of high spatial and temporal precision are important requisites for studying the real-time dynamics of neural networks. Multiple shank silicon probes enable large-scale monitoring of individual neurons. Optical stimulation of genetically targeted neurons expressing light sensitive channels or other fast (milliseconds) actuators offers the means for controlled perturbation of local circuits. Here we describe a method to equip the shanks of silicon probes with micron-scale light guides for allowing the simultaneous use of both approaches. We then show illustrative examples of how these compact hybrid electrodes can be used in probing local circuits in behaving rats and mice. A key advantage of these devices is the enhanced spatial precision of stimulation that is achieved by delivering light close to the recording sites of the probe. When paired with the expression of light sensitive actuators within genetically specified neuronal populations, these devices allow the relatively straightforward and interpretable manipulation of network activity.
doi:10.1111/j.1460-9568.2010.07250.x
PMCID: PMC2954764  PMID: 20529127
circuit analysis; optrode; ChR2; halorhodopsin; excitation; inhibition
19.  Fabrication of Pillar Shaped Electrode Arrays for Artificial Retinal Implants 
Sensors (Basel, Switzerland)  2008;8(9):5845-5856.
Polyimide has been widely applied to neural prosthetic devices, such as the retinal implants, due to its well-known biocompatibility and ability to be micropatterned. However, planar films of polyimide that are typically employed show a limited ability in reducing the distance between electrodes and targeting cell layers, which limits site resolution for effective multi-channel stimulation. In this paper, we report a newly designed device with a pillar structure that more effectively interfaces with the target. Electrode arrays were successfully fabricated and safely implanted inside the rabbit eye in suprachoroidal space. Optical Coherence Tomography (OCT) showed well-preserved pillar structures of the electrode without damage. Bipolar stimulation was applied through paired sites (6:1) and the neural responses were successfully recorded from several regions in the visual cortex. Electrically evoked cortical potential by the pillar electrode array stimulation were compared to visual evoked potential under full-field light stimulation.
doi:10.3390/s8095845
PMCID: PMC3705534
Polyimide; retinal implant; pillar; optical coherence tomography; electrically evoked cortical potential
20.  Implantable microelectrode arrays for simultaneous electrophysiological and neurochemical recordings 
Journal of neuroscience methods  2008;174(1):62-70.
Implantable microfabricated microelectrode arrays represent a versatile and powerful tool to record electrophysiological activity across multiple spatial locations in the brain. Spikes and field potentials, however, correspond to only a fraction of the physiological information available at the neural interface. In urethane-anesthetized rats, microfabricated microelectrode arrays were implanted acutely for simultaneous recording of striatal local field potentials, spikes, and electrically-evoked dopamine overflow on the same spatiotemporal scale. During these multi-modal recordings we observed 1) that the amperometric method used to detect dopamine did not significantly influence electrophysiological activity, 2) that electrical stimulation in the medial forebrain bundle (MFB) region resulted in electrochemically-transduced dopamine transients in the striatum that were spatially heterogeneous within at least 200 µm, and 3) following MFB stimulation, dopamine levels and electrophysiological activity within the striatum exhibited similar temporal profiles. These neural probes are capable of incorporating customized microelectrode geometries and configurations, which may be useful for examining specific spatiotemporal relationships between electrical and chemical signaling in the brain.
doi:10.1016/j.jneumeth.2008.06.036
PMCID: PMC2743487  PMID: 18692090
neural probe; amperometry; dopamine; spike recordings; local field potentials; striatum
21.  Delivery of continuously-varying stimuli using channelrhodopsin-2 
To study sensory processing, stimuli are delivered to the sensory organs of animals and evoked neural activity is recorded downstream. However, noise and uncontrolled modulatory input can interfere with repeatable delivery of sensory stimuli to higher brain regions. Here we show how channelrhodopsin-2 (ChR2) can be used to deliver continuous, subthreshold, time-varying currents to neurons at any point along the sensory-motor pathway. To do this, we first deduce the frequency response function of ChR2 using a Markov model of channel kinetics. We then confirm ChR2's frequency response characteristics using continuously-varying optical stimulation of neurons that express one of three ChR2 variants. We find that wild-type ChR2 and the E123T/H134R mutant (“ChETA”) can pass continuously-varying subthreshold stimuli with frequencies up to ~70 Hz. Additionally, we find that wild-type ChR2 exhibits a strong resonance at ~6–10 Hz. Together, these results indicate that ChR2-derived optogenetic tools are useful for delivering highly repeatable artificial stimuli that mimic in vivo synaptic bombardment.
doi:10.3389/fncir.2013.00184
PMCID: PMC3853882  PMID: 24367294
channelrhodopsin-2; linear response theory; dynamical systems; neural circuits; networks and dynamical systems; circuit dynamics; optogenetics; electrophysiology methods
22.  Optogenetic stimulation effectively enhances intrinsically generated network synchrony 
Synchronized bursting is found in many brain areas and has also been implicated in the pathophysiology of neuropsychiatric disorders such as epilepsy, Parkinson’s disease, and schizophrenia. Despite extensive studies of network burst synchronization, it is insufficiently understood how this type of network wide synchronization can be strengthened, reduced, or even abolished. We combined electrical recording using multi-electrode array with optical stimulation of cultured channelrhodopsin-2 transducted hippocampal neurons to study and manipulate network burst synchronization. We found low frequency photo-stimulation protocols that are sufficient to induce potentiation of network bursting, modifying bursting dynamics, and increasing interneuronal synchronization. Surprisingly, slowly fading-in light stimulation, which substantially delayed and reduced light-driven spiking, was at least as effective in reorganizing network dynamics as much stronger pulsed light stimulation. Our study shows that mild stimulation protocols that do not enforce particular activity patterns onto the network can be highly effective inducers of network-level plasticity.
doi:10.3389/fncir.2013.00167
PMCID: PMC3805139  PMID: 24155695
optogenetics; multi-electrode arrays; network-level plasticity; bursting; synchronization
23.  Intraocular retinal prosthesis. 
PURPOSE: An electronic implant that can bypass the damaged photoreceptors and electrically stimulate the remaining retinal neurons to restore useful vision has been proposed. A number of key questions remain to make this approach feasible. The goal of this thesis is to address the following 2 specific null hypotheses: (1) Stimulus parameters make no difference in the electrically elicited retinal responses. (2) Just as we have millions of photoreceptors, so it will take a device that can generate millions of pixels/light points to create useful vision. METHODS: For electrophysiologic experiments, 2 different setups were used. In the first setup, charge-balanced pulses were delivered to the retinal surface via electrodes inserted through an open sky approach in normal or blind retinal degenerate (rd) mice. In the second setup, the rabbit retina was removed under red light conditions from an enucleated eye and then maintained in a chamber while being superfused with oxygenated, heated Ames media. In both setups, stimulating electrodes and recording electrodes were positioned on the retinal surface to evaluate the effect of varying stimulation parameters on the orthodromic retinal responses (i.e., recording electrode placed between stimulating electrodes and optic nerve head). For psychophysical experiments, visual images were divided into pixels of light that could be projected in a pattern on the retina in up to 8 sighted volunteers. Subjects were asked to perform various tasks ranging from reading and face recognition to various activities of daily living. RESULTS: Electrophysiologic experiments: In a normal mouse, a single cycle of a 1-kHz sine wave was significantly more efficient than a 1-kHz square wave (P < .05), but no such difference was noted in either of the 8- or 16-week-old rd mouse groups (8-week-old, P = .426; 16-week-old, P = .078). Charge threshold was significantly higher in 16-week-old rd mouse versus both 8-week-old rd and normal mouse for every stimulus duration (P < .05). In all groups, short duration pulses (40, 80, and 120 microseconds) were more efficient in terms of total charge (the product of pulse amplitude and pulse duration) than longer (500 and 1,000 microseconds) pulses (P < .05). In all groups, applying a pulse train did not lead to more efficient charge usage (P < .05). Psychophysical experiments: In high-contrast tests, facial recognition rates of over 75% were achieved for all subjects with dot sizes of up to 31.5 minutes of arc when using a 25 x 25 grid with 4.5 arc minute gaps, a 30% dropout rate, and 6 gray levels. Even with a 4 x 4 array of pixels, some subjects were able to accurately describe 2 of the objects. Subjects who were able to read the 4-pixel letter height sentences (on the 6 x 10 and 16 x 16 array) seemed to have a good scanning technique. Scanning at the proper velocity tends to bring out more contrast in the lettering. The reading speed for the 72-point font is a bit slower than for the next smaller font. This may be due to the limited number of letters (3) visible in the window with this large font. CONCLUSIONS: Specific parameters needed to stimulate the retina were identified. Delineating the optimum parameters will decrease the current requirements. Psychophysical tests show that with limited pixels and image processing, useful vision is possible. Both these findings should greatly simplify the engineering of an electronic retinal prosthesis.
PMCID: PMC1359018  PMID: 11797315
24.  Optical control of neuronal excitation and inhibition using a single opsin protein, ChR2 
Scientific Reports  2013;3:3110.
The effect of electrical stimulation on neuronal membrane potential is frequency dependent. Low frequency electrical stimulation can evoke action potentials, whereas high frequency stimulation can inhibit action potential transmission. Optical stimulation of channelrhodopsin-2 (ChR2) expressed in neuronal membranes can also excite action potentials. However, it is unknown whether optical stimulation of ChR2-expressing neurons produces a transition from excitation to inhibition with increasing light pulse frequencies. Here we report optical inhibition of motor neuron and muscle activity in vivo in the cooled sciatic nerves of Thy1-ChR2-EYFP mice. We also demonstrate all-optical single-wavelength control of neuronal excitation and inhibition without co-expression of inhibitory and excitatory opsins. This all-optical system is free from stimulation-induced electrical artifacts and thus provides a new approach to investigate mechanisms of high frequency inhibition in neuronal circuits in vivo and in vitro.
doi:10.1038/srep03110
PMCID: PMC3813941  PMID: 24173561
25.  Holographic Photolysis for Multiple Cell Stimulation in Mouse Hippocampal Slices 
PLoS ONE  2010;5(2):e9431.
Background
Advanced light microscopy offers sensitive and non-invasive means to image neural activity and to control signaling with photolysable molecules and, recently, light-gated channels. These approaches require precise and yet flexible light excitation patterns. For synchronous stimulation of subsets of cells, they also require large excitation areas with millisecond and micrometric resolution. We have recently developed a new method for such optical control using a phase holographic modulation of optical wave-fronts, which minimizes power loss, enables rapid switching between excitation patterns, and allows a true 3D sculpting of the excitation volumes. In previous studies we have used holographic photololysis to control glutamate uncaging on single neuronal cells. Here, we extend the use of holographic photolysis for the excitation of multiple neurons and of glial cells.
Methods/Principal Findings
The system combines a liquid crystal device for holographic patterned photostimulation, high-resolution optical imaging, the HiLo microscopy, to define the stimulated regions and a conventional Ca2+ imaging system to detect neural activity. By means of electrophysiological recordings and calcium imaging in acute hippocampal slices, we show that the use of excitation patterns precisely tailored to the shape of multiple neuronal somata represents a very efficient way for the simultaneous excitation of a group of neurons. In addition, we demonstrate that fast shaped illumination patterns also induce reliable responses in single glial cells.
Conclusions/Significance
We show that the main advantage of holographic illumination is that it allows for an efficient excitation of multiple cells with a spatiotemporal resolution unachievable with other existing approaches. Although this paper focuses on the photoactivation of caged molecules, our approach will surely prove very efficient for other probes, such as light-gated channels, genetically encoded photoactivatable proteins, photoactivatable fluorescent proteins, and voltage-sensitive dyes.
doi:10.1371/journal.pone.0009431
PMCID: PMC2828488  PMID: 20195547

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