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1.  A Coaxial Optrode As Multifunction Write-Read Probe for Optogenetic Studies in Non-Human Primates 
Journal of neuroscience methods  2013;219(1):142-154.
Background
Advances in optogenetics have led to first reports of expression of light-gated ion-channels in non-human primates (NHPs). However, a major obstacle preventing effective application of optogenetics in NHPs and translation to optogenetic therapeutics is the absence of compatible multifunction optoelectronic probes for (1) precision light delivery, (2) low-interference electrophysiology, (3) protein fluorescence detection, and (4) repeated insertion with minimal brain trauma.
New Method
Here we describe a novel brain probe device, a “coaxial optrode”, designed to minimize brain tissue damage while microfabricated to perform simultaneous electrophysiology, light delivery and fluorescence measurements in the NHP brain. The device consists of a tapered, gold-coated optical fiber inserted in a polyamide tube. A portion of the gold coating is exposed at the fiber tip to allow electrophysiological recordings in addition to light delivery/collection at the tip.
Results
Coaxial optrode performance was demonstrated by experiments in rodents and NHPs, and characterized by computational models. The device mapped opsin expression in the brain and achieved precisely targeted optical stimulation and electrophysiology with minimal cortical damage.
Comparison with Existing Methods
Overall, combined electrical, optical and mechanical features of the coaxial optrode allowed a performance for NHP studies which was not possible with previously existing devices.
Conclusions
Coaxial optrode is currently being used in two NHP laboratories as a major tool to study brain function by inducing light modulated neural activity and behavior. By virtue of its design, the coaxial optrode can be extended for use as a chronic implant and multisite neural stimulation/recording.
doi:10.1016/j.jneumeth.2013.06.011
PMCID: PMC3789534  PMID: 23867081
optogenetics; optoelectronic devices; non-human primates; fluorescence detection; tissue heating; light propagation in tissue
2.  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
3.  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
4.  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
5.  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
6.  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
7.  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
8.  Arrays of MicroLEDs and Astrocytes: Biological Amplifiers to Optogenetically Modulate Neuronal Networks Reducing Light Requirement 
PLoS ONE  2014;9(9):e108689.
In the modern view of synaptic transmission, astrocytes are no longer confined to the role of merely supportive cells. Although they do not generate action potentials, they nonetheless exhibit electrical activity and can influence surrounding neurons through gliotransmitter release. In this work, we explored whether optogenetic activation of glial cells could act as an amplification mechanism to optical neural stimulation via gliotransmission to the neural network. We studied the modulation of gliotransmission by selective photo-activation of channelrhodopsin-2 (ChR2) and by means of a matrix of individually addressable super-bright microLEDs (μLEDs) with an excitation peak at 470 nm. We combined Ca2+ imaging techniques and concurrent patch-clamp electrophysiology to obtain subsequent glia/neural activity. First, we tested the μLEDs efficacy in stimulating ChR2-transfected astrocyte. ChR2-induced astrocytic current did not desensitize overtime, and was linearly increased and prolonged by increasing μLED irradiance in terms of intensity and surface illumination. Subsequently, ChR2 astrocytic stimulation by broad-field LED illumination with the same spectral profile, increased both glial cells and neuronal calcium transient frequency and sEPSCs suggesting that few ChR2-transfected astrocytes were able to excite surrounding not-ChR2-transfected astrocytes and neurons. Finally, by using the μLEDs array to selectively light stimulate ChR2 positive astrocytes we were able to increase the synaptic activity of single neurons surrounding it. In conclusion, ChR2-transfected astrocytes and μLEDs system were shown to be an amplifier of synaptic activity in mixed corticalneuronal and glial cells culture.
doi:10.1371/journal.pone.0108689
PMCID: PMC4180921  PMID: 25265500
9.  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
10.  An implantable neural probe with monolithically integrated dielectric waveguide and recording electrodes for optogenetics applications 
Journal of neural engineering  2013;10(5):056012.
Objective
Optogenetics promises exciting neuroscience research by offering optical stimulation of neurons with unprecedented temporal resolution, cell-type specificity and the ability to excite as well as to silence neurons. This work provides the technical solution to deliver light to local neurons and record neural potentials, facilitating local circuit analysis and bridging the gap between optogenetics and neurophysiology research.
Approach
We have designed and obtained the first in vivo validation of a neural probe with monolithically integrated electrodes and waveguide. High spatial precision enables optical excitation of targeted neurons with minimal power and recording of single-units in dense cortical and subcortical regions.
Main results
The total coupling and transmission loss through the dielectric waveguide at 473 nm was 10.5 ± 1.9 dB, corresponding to an average output intensity of 9400 mW mm−2 when coupled to a 7 mW optical fiber. Spontaneous field potentials and spiking activities of multiple Channelrhodopsin-2 expressing neurons were recorded in the hippocampus CA1 region of an anesthetized rat. Blue light stimulation at intensity of 51 mW mm−2 induced robust spiking activities in the physiologically identified local populations.
Significance
This minimally invasive, complete monolithic integration provides unmatched spatial precision and scalability for future optogenetics studies at deep brain regions with high neuronal density.
doi:10.1088/1741-2560/10/5/056012
PMCID: PMC4056669  PMID: 23985803
11.  Optical mapping of optogenetically shaped cardiac action potentials 
Scientific Reports  2014;4:6125.
Light-mediated silencing and stimulation of cardiac excitability, an important complement to electrical stimulation, promises important discoveries and therapies. To date, cardiac optogenetics has been studied with patch-clamp, multielectrode arrays, video microscopy, and an all-optical system measuring calcium transients. The future lies in achieving simultaneous optical acquisition of excitability signals and optogenetic control, both with high spatio-temporal resolution. Here, we make progress by combining optical mapping of action potentials with concurrent activation of channelrhodopsin-2 (ChR2) or halorhodopsin (eNpHR3.0), via an all-optical system applied to monolayers of neonatal rat ventricular myocytes (NRVM). Additionally, we explore the capability of ChR2 and eNpHR3.0 to shape action-potential waveforms, potentially aiding the study of short/long QT syndromes that result from abnormal changes in action potential duration (APD). These results show the promise of an all-optical system to acquire action potentials with precise temporal optogenetics control, achieving a long-sought flexibility beyond the means of conventional electrical stimulation.
doi:10.1038/srep06125
PMCID: PMC4137261  PMID: 25135113
12.  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
13.  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
14.  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
15.  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
16.  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
17.  A CMOS IC-based multisite measuring system for stimulation and recording in neural preparations in vitro 
In this report, we describe the system integration of a complementary metal oxide semiconductor (CMOS) integrated circuit (IC) chip, capable of both stimulation and recording of neurons or neural tissues, to investigate electrical signal propagation within cellular networks in vitro. The overall system consisted of three major subunits: a 5.0 × 5.0 mm CMOS IC chip, a reconfigurable logic device (field-programmable gate array, FPGA), and a PC. To test the system, microelectrode arrays (MEAs) were used to extracellularly measure the activity of cultured rat cortical neurons and mouse cortical slices. The MEA had 64 bidirectional (stimulation and recording) electrodes. In addition, the CMOS IC chip was equipped with dedicated analog filters, amplification stages, and a stimulation buffer. Signals from the electrodes were sampled at 15.6 kHz with 16-bit resolution. The measured input-referred circuitry noise was 10.1 μ V root mean square (10 Hz to 100 kHz), which allowed reliable detection of neural signals ranging from several millivolts down to approximately 33 μ Vpp. Experiments were performed involving the stimulation of neurons with several spatiotemporal patterns and the recording of the triggered activity. An advantage over current MEAs, as demonstrated by our experiments, includes the ability to stimulate (voltage stimulation, 5-bit resolution) spatiotemporal patterns in arbitrary subsets of electrodes. Furthermore, the fast stimulation reset mechanism allowed us to record neuronal signals from a stimulating electrode around 3 ms after stimulation. We demonstrate that the system can be directly applied to, for example, auditory neural prostheses in conjunction with an acoustic sensor and a sound processing system.
doi:10.3389/fneng.2014.00039
PMCID: PMC4193337  PMID: 25346683
bidirectional electrode; CMOS IC technology; low-noise amplifier; neural networks; noise suppressor
18.  A system for optically controlling neural circuits with very high spatial and temporal resolution 
Optogenetics offers a powerful new approach for controlling neural circuits. It has a vast array of applications in both basic and clinical science. For basic science, it opens the door to unraveling circuit operations, since one can perturb specific circuit components with high spatial (single cell) and high temporal (millisecond) resolution. For clinical applications, it allows new kinds of selective treatments, because it provides a method to inactivate or activate specific components in a malfunctioning circuit and bring it back into a normal operating range [1–3]. To harness the power of optogenetics, though, one needs stimulating tools that work with the same high spatial and temporal resolution as the molecules themselves, the channelrhodopsins. To date, most stimulating tools require a tradeoff between spatial and temporal precision and are prohibitively expensive to integrate into a stimulating/recording setup in a laboratory or a device in a clinical setting [4, 5]. Here we describe a Digital Light Processing (DLP)-based system capable of extremely high temporal resolution (sub-millisecond), without sacrificing spatial resolution. Furthermore, it is constructed using off-the-shelf components, making it feasible for a broad range of biology and bioengineering labs. Using transgenic mice that express channelrhodopsin-2 (ChR2), we demonstrate the system’s capability for stimulating channelrhodopsin-expressing neurons in tissue with single cell and sub-millisecond precision.
PMCID: PMC4331115
19.  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
20.  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
21.  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
22.  Graphene-based carbon-layered electrode array technology for neural imaging and optogenetic applications 
Nature Communications  2014;5:5258.
Neural micro-electrode arrays that are transparent over a broad wavelength spectrum from ultraviolet to infrared could allow for simultaneous electrophysiology and optical imaging, as well as optogenetic modulation of the underlying brain tissue. The long-term biocompatibility and reliability of neural micro-electrodes also require their mechanical flexibility and compliance with soft tissues. Here we present a graphene-based, carbon-layered electrode array (CLEAR) device, which can be implanted on the brain surface in rodents for high-resolution neurophysiological recording. We characterize optical transparency of the device at >90% transmission over the ultraviolet to infrared spectrum and demonstrate its utility through optical interface experiments that use this broad spectrum transparency. These include optogenetic activation of focal cortical areas directly beneath electrodes, in vivo imaging of the cortical vasculature via fluorescence microscopy and 3D optical coherence tomography. This study demonstrates an array of interfacing abilities of the CLEAR device and its utility for neural applications.
Monitoring neuronal activity in the rodent in vivo brain is commonly done using micro-electrode arrays but these devices are not normally compatible with optical technologies. Here the authors design a transparent and flexible electrode array based on graphene that allows them to combine electrophysiological recordings with optogenetic and imaging experiments.
doi:10.1038/ncomms6258
PMCID: PMC4218963  PMID: 25327513
23.  Achieving High-Frequency Optical Control of Synaptic Transmission 
The Journal of Neuroscience  2014;34(22):7704-7714.
The optogenetic tool channelrhodopsin-2 (ChR2) is widely used to excite neurons to study neural circuits. Previous optogenetic studies of synapses suggest that light-evoked synaptic responses often exhibit artificial synaptic depression, which has been attributed to either the inability of ChR2 to reliably fire presynaptic axons or to ChR2 elevating the probability of release by depolarizing presynaptic boutons. Here, we compare light-evoked and electrically evoked synaptic responses for high-frequency stimulation at three synapses in the mouse brain. At synapses from Purkinje cells to deep cerebellar nuclei neurons (PC→DCN), light- and electrically evoked synaptic currents were remarkably similar for ChR2 expressed transgenically or with adeno-associated virus (AAV) expression vectors. For hippocampal CA3→CA1 synapses, AAV expression vectors of serotype 1, 5, and 8 led to light-evoked synaptic currents that depressed much more than electrically evoked currents, even though ChR2 could fire axons reliably at up to 50 Hz. The disparity between optical and electrical stimulation was eliminated when ChR2 was expressed transgenically or with AAV9. For cerebellar granule cell to stellate cell (grc→SC) synapses, AAV1 also led to artificial synaptic depression and AAV9 provided superior performance. Artificial synaptic depression also occurred when stimulating over presynaptic boutons, rather than axons, at CA3→CA1 synapses, but not at PC→DCN synapses. These findings indicate that ChR2 expression methods and light stimulation techniques influence synaptic responses in a neuron-specific manner. They also identify pitfalls associated with using ChR2 to study synapses and suggest an approach that allows optogenetics to be applied in a manner that helps to avoid potential complications.
doi:10.1523/JNEUROSCI.4694-13.2014
PMCID: PMC4035530  PMID: 24872574
AAV; channelrhodopsin; optogenetics; short-term plasticity; synapse
24.  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
25.  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

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