Scanning small spots of laser light allows mapping of synaptic circuits in brain slices from transgenic mice expressing channelrhodopsin-2 (ChR2). These light spots photostimulate presynaptic neurons expressing ChR2, while postsynaptic responses can be monitored in neurons that do not express ChR2. Correlating the location of the light spot with the amplitude of the postsynaptic response elicited at that location yields maps of the spatial organization of the synaptic circuits. This approach yields maps within minutes, which is several orders of magnitude faster than can be achieved with conventional paired electrophysiological methods. We have applied this high-speed technique to map local circuits in many brain regions. In cerebral cortex, we observed that maps of excitatory inputs to pyramidal cells were qualitatively different from those measured for interneurons within the same layers of the cortex. In cerebellum, we have used this approach to quantify the convergence of molecular layer interneurons on to Purkinje cells. The number of converging interneurons is reduced by treatment with gap junction blockers, indicating that electrical synapses between interneurons contribute substantially to the spatial convergence. Remarkably, gap junction blockers affect convergence in sagittal cerebellar slices but not in coronal slices, indicating sagittal polarization of electrical coupling between interneurons. By measuring limb movement or other forms of behavioral output, this approach also can be used in vivo to map brain circuits non-invasively. In summary, ChR2-mediated high-speed mapping promises to revolutionize our understanding of brain circuitry.

Here, we introduce a computational procedure to examine whether optogenetically activated neuronal firing recordings could be characterized as multifractal series. Optogenetics is emerging as a valuable experimental tool and a promising approach for studying a variety of neurological disorders in animal models. The spiking patterns from cortical region of the brain of optogenetically-stimulated transgenic mice were analyzed using a sophisticated fluctuation analysis method known as multifractal detrended fluctuation analysis (MFDFA). We observed that the optogenetically-stimulated neural firings are consistent with a multifractal process. Further, we used MFDFA to monitor the effect of chemically induced pain (formalin injection) and optogenetic treatment used to relieve the pain. In this case, dramatic changes in parameters characterizing a multifractal series were observed. Both the generalized Hurst exponent and width of singularity spectrum effectively differentiates the neural activities during control and pain induction phases. The quantitative nature of the analysis equips us with better measures to quantify pain. Further, it provided a measure for effectiveness of the optogenetic stimulation in inhibiting pain. MFDFA-analysis of spiking data from other deep regions of the brain also turned out to be multifractal in nature, with subtle differences in the parameters during pain-induction by formalin injection and inhibition by optogenetic stimulation. Characterization of neuronal firing patterns using MFDFA will lead to better understanding of neuronal response to optogenetic activation and overall circuitry involved in the process.

Optogenetics has proven to be a powerful tool for understanding the function of specific cell types and circuits within the central nervous system and establishing a causal link between their activity and behavior. Its application in non-human primates has been slow to develop. One challenge has been the damage caused by transdural delivery of viruses and light to the brain. Here, we report optogenetic activation of neuronal responses in the alert and behaving monkey after replacement of the native dura with a transparent artificial dura. This approach enables the use of fine glass micropipettes to inject virus with minimal damage and transdural illumination, obviating the damage that would otherwise occur as a result of lowering optical fibers into the brain. It also permits visualization of the underlying cortical micro-vasculature, which has proven to be helpful in targeting electrodes and laser illumination to the virus location. This approach promises to greatly assist in the dissection of cortical circuits underlying visual perception and behavior.

The habenula is a brain region found in all vertebrate species. It consists of medial and lateral subnuclei which make complex descending connections to the brainstem. Although the medial habenula (MHb) and its projection, the fasciculus retroflexus (FR), have been recognized for decades, their function remains obscure. The small size of the MHb in rodents, and the cellular and molecular complexity of this region, have made it difficult to study the function of this region with high specificity. Here we describe a Cre-mediated genetic system for expressing the microbial opsin channelrhodopsin (ChR2) specifically in the dorsal (dMHb) and ventral (vMHb) medial habenula. Genetically targeted expression of ChR2 allows MHb neurons to be selectively activated with light in acute brain slices with electrophysiological readouts, and in vivo by means of custom-built fiber optic cannulas. These tools will allow highly specific studies of MHb circuitry and the role of the MHb in behaviors related to addiction and mood regulation.

We employed a transgenic mouse having conditional expression of ChR2(H134R) in neurons of the inferior olive to facilitate understanding of the role of electrical coupling and oscillation in central nervous system function. Two-photon excitation of ChR2-expressing neurons using 64 laser beams restricted to single inferior olive cell bodies depolarized neurons and evoked voltage deflections in neighboring neurons demonstrating electrical coupling. Broader illumination of neuronal ensembles using blue light induced an optical clamp of endogenous electrical rhythms in the inferior olive of acutely-prepared brain slices, which when applied in vivo directly modulated the local field potential activity and induced tremor. The experiments demonstrate novel methods to optically manipulate electrically coupled potentials and rhythmogenesis within a neuronal ensemble. From a functional perspective, the experiments shed light on the cellular and circuitry mechanisms of essential tremor, a prevalent neurological condition, by indicating time- and frequencydependence of tremor upon varying rhythms of inferior olive stimulation. The experiments indicate analog control of a brain rhythm that may be used to enhance our understanding of the functional consequences of central rhythmogenesis.

As a critical basis of functional brain imaging, neurovascular coupling describes the link between neuronal and hemodynamic changes. The majority of in vivo neurovascular coupling studies was performed by inducing sensory stimulation via afferent inputs. Unfortunately such an approach results in recruiting of multiple types of cells, which confounds the explanation of neuronal roles in stimulus evoked hemodynamic changes. Recently optogenetics has emerged to provide immediate control of neurons by exciting or inhibiting genetically engineered neurons expressing light sensitive proteins. However, there is a need for optical methods capable of imaging the concurrent hemodynamic changes. We utilize laser speckle contrast imaging (LSCI) to obtain high resolution display of cerebral blood flow (CBF) in the vicinity of the targeted neural population. LSCI is a minimally invasive method for imaging CBF in microvessels through thinned skull, and produces images with high spatiotemporal resolution, wide field of view. In the integrated system light sources with different wavelengths and band-passing/blocking filters were used to allow simultaneous optical manipulation of neuronal activities and optical imaging of corresponding CBF. Experimental studies were carried out in a rodent model expressing channalrhodopsin (ChR2) in excitatory neurons in the somatosensory cortex (S1). The results demonstrated significant increases of CBF in response to ChR2 stimulation (exciting neuronal firing) comparable to the CBF response to contralateral forepaw stimulation. The approach promises to be an exciting minimally invasive method to study neurovascular coupling. The complete system provides a novel approach for broad neuroscience applications.

Optogenetics technology has opened new landscapes for neuroscience research. Due to its non-diffracting and selfhealing nature, Bessel beam has potential to improve in-depth optogenetic stimulation. A detailed understanding of Bessel beam propagation, as well as its superiority over commonly used Gaussian beam, is essential for delivery and control of light irradiation for optogenetics and other light stimulation approaches. We developed an algorithm for modeling Bessel beam propagation and then compared both beam propagations in two-layered mice brain under variance of multiple variables (i.e., wavelength, numerical aperture, and beam size). These simulations show that Bessel beam is significantly advantageous over Gaussian beam for in-depth optogenetic stimulation, leading to development of lessinvasive probes. While experimental measurements using single-photon Bessel-Gauss beam generated by axicon-tip fiber did not show improved stimulation-depth, near-infrared Bessel beam generated using free-space optics and an axicon led to better penetration than near-infrared Gaussian beam.

Outer hair cell (OHC) is widely accepted as the origin of cochlear amplification, a mechanism that accounts for the extreme sensitivity of the mammalian hearing. The key process of cochlear amplification is the reverse transduction, where the OHC changes its length under electrical stimulation. In this study, we developed a method to modulate electro-mechanical transduction with an optogenetic approach based on channelrhodopsin 2 (ChR2), a direct lightactivated non-selective cation channel (NSCC). We specifically expressed ChR2 in mouse cochlea OHCs through in uterus injection of adenovirus vector with ChR2 in fusion with the fluorescent marker tdTomato. We also transfected ChR2(H134R), a point mutant of ChR2, with plasmid to an auditory cell line (HEI-OC1). With whole cell recording, we found that blue light (470 nm) elicited a current with a reversal potential around zero in both mouse OHCs and HEI-OC1 cells and generated depolarization in both cell types.

Controlled release of ATP can be used for understanding extracellular purinergic signaling. While coarse mechanical forces and hypotonic stimulation have been utilized in the past to initiate ATP release from cells, these methods are neither spatially accurate nor temporally precise. Further, these methods cannot be utilized in a highly effective cell-specific manner. To mitigate the uncertainties regarding cellular-specificity and spatio-temporal release of ATP, we herein demonstrate use of optogenetics for ATP release. ATP release in response to optogenetic stimulation was monitored by Luciferin-Luciferase assay (North American firefly, photinus pyralis) using luminometer as well as mesoscopic bioluminescence imaging. Our result demonstrates repetitive release of ATP subsequent to optogenetic stimulation. It is thus feasible that purinergic signaling can be directly detected via imaging if the stimulus can be confined to single cell or in a spatially-defined group of cells. This study opens up new avenue to interrogate the mechanisms of purinergic signaling.

The breakthrough discovery of a nanoscale optically gated ion channel protein, Channelrhodopsin 2 (ChR2), and its combination with a genetically expressed ion pump, Halorhodopsin, allowed the direct stimulation and inhibition of individual action potentials with light alone. This work reports developments of ultra-bright elec­ tronically controlled optical array sources with enhanced light gated ion channels and pumps for use in systems to further our understanding of both brain and visual function. This work is undertaken as part of the European project, OptoNeuro. Micro-LED arrays permit spatio-temporal control of neuron stimulation on sub-millisecond timescales. However they are disadvantaged by their broad spatial light emission distribution and low fill factor. We present the design and implementation of a projection and micro-optics system for use with a micro-LED array consisting of a 16x16 matrix of 25 μm diameter micro-LEDs with 150 μm centre-to-centre spacing and an emission spectrum centred at 470 nm overlapping the peak sensitivity of ChR2 and its testing on biological samples. The projection system images the micro-LED array onto micro-optics to improve the fill-factor from ~2% to more than 78% by capturing a larger fraction of the LED emission and directing it correctly to the sample plane. This approach allows low fill factor arrays to be used effectively, which in turn has benefits in terms of thermal management and electrical drive from CMOS backplane electronics. The entire projection system is integrated into a microscope prototype to provide stimulation spots at the same size as the neuron cell body (μ10 pm).

Numerous studies in Alzheimer’s Disease (AD) animal models show that overproduction of Aβ peptides and their oligomerization can distort dendrites, damage synapses, and decrease the number of dendritic spines and synapses. Aβ may trigger synapse loss by modulating activity of actin-regulating proteins, such as Rac1 and cofilin. Indeed, Aβ1-42 oligomers can activate actin severing protein cofilin through calcineurin-mediated activation of phosphatase slingshot and inhibit an opposing pathway that suppresses cofilin phosphorylation through Rac-mediated activation of LIMK1. Excessive activation of actin-severing protein cofilin triggers the formation of a non-dynamic actin bundles, called rods that are found in AD brains and cause loss of synapses. Hence, regulation of these actin-regulating proteins in dendritic spines could potentially provide useful tools for preventing the synapse/spine loss associated with earlier stages of AD neuropathology. However, lack of spatiotemporal control over their activity is a key limitation. Recently, optogenetic advancements have provided researchers with convenient light-activating proteins such as photoactivatable Rac (PARac). Here, we transfected cultured primary hippocampal neurons and human embryonic kidney (HEK) cells with a PARac/ mCherry-containing plasmid and the mCherry-positive cells were identified and imaged using an inverted fluorescence microscope. Rac1 activation was achieved by irradiation with blue light (480nm) and live changes in dendritic spine morphology were observed using mCherry (587nm). Rac activation was confirmed by immunostaining for phosphorylated form of effector proteinP21 protein-activated kinase 1 (PAK1) and reorganization of actin. Thus, our studies confirm the feasibility of using the PA-Rac construct to trigger actin re-organization in the dendritic spines.

Significant efforts are being made for control on axonal guidance due to its importance in nerve regeneration and in the formation of functional neuronal circuitry in-vitro. These include several physical (topographic modification, optical force, and electric field), chemical (surface functionalization cues) and hybrid (electro-chemical, photochemical etc) methods. Here, we report comparison of the effect of linear flow versus microfluidic flow produced by an opticallydriven micromotor in guiding retinal ganglion axons. A circularly polarized laser tweezers was used to hold, position and spin birefringent calcite particle near growth cone, which in turn resulted in microfluidic flow. The flow rate and resulting shear-force on axons could be controlled by a varying the power of the laser tweezers beam. The calcite particles were placed separately in one chamber and single particle was transported through microfluidic channel to another chamber containing the retina explant. In presence of flow, the turning of axons was found to strongly correlate with the direction of flow. Turning angle as high as 90° was achieved. Optofluidic-manipulation can be applied to other types of mammalian neurons and also can be extended to stimulate mechano-sensing neurons.

The ability to control the illumination and imaging paths of optical microscopes is an essential part of advanced fluorescence microscopy, and a powerful tool for optogenetics. In order to maximize the visualization and the image quality of the objects under observation we use programmable, fast Micro Mirror Arrays (MMAs) as high-resolution Spatial Light Modulators (SLMs). Using two 256x256 MMAs in a mirror-based illumination setup allows for fast angular-spatial control at a wide range of wavelengths (300-1000nm). Additionally, the illumination intensity can be controlled at 10-bit resolution. The setup allows selective illumination of subcellular regions of interest enabling the precise, localized activation of fluorescent probes and the activation and deactivation of subcellular and cellular signaling cascades using photo-activated ion-channels. Furthermore, inasmuch as phototoxicity is dependent on the rate of photo illumination [1] we show that our system, which provides fast, compartmentalized illumination is minimally phototoxic.

Early-generation penetrating waveguide arrays made of glass (SiO2) were micromachined for optogenetic stimulation in able to provide comprehensive and selective access to distributed targets in three dimensions. We characterized light delivery of the device in order to facilitate design optimization and understand its application in tissue. The glass optrodes were formed by dicing, etching, and annealing. A fused silica/quartz substrate was used to produce 10×10 arrays of optrodes with constant geometry having a pyramidal tip at the end of a straight-edge shank; length, width, spacing, tip angle, and even array size can be varied indepedently. Visible light transmission effciency of optrodes was investigated with input from an optical fiber as well as microscope objective lenses. With a 120-µm wide and 1.5-mm long optrode having a tip taper angle of 30º with respect to the optical axis, almost 90% of visible and IR light from a butt-coupled 50-µm multimode fiber is transmitted out of the optrode tips when optrode shanks are surrounded by tissue. In air, the normalized output power decreases according to the area mismatch betweeen optrode shank and the focused beam width from the microscope; visible light transmission is as much as 90% as well.

Recently developed optogenetics techniques have enabled researchers to modulate the activity of specific cell types. As a result, complex neural pathways previously regarded as black boxes can now be directly probed, yielding a steadily increasing understanding of the basic neural circuits that underlie health and disease. For in vivo experimentation, fiber-coupled lasers have traditionally been used to illuminate internal brain regions, via an optical fiber that penetrates through overlying tissue. Though able to deliver intense fiber-coupled light, lasers are costly, bulky, and face limitations in output beam stability and temporal precision during modulated outputs. For experiments on unrestricted, behaving animals, a laser-based system also necessitates the use of fiber optic rotary joints, which come with costs and limitations of their own. Here, we report and characterize an alternative light delivery solution, based on high intensity fiber-coupled LEDs that are miniaturized for placement on the end of custom electrical commutators. This design allows for enhanced control of output light and expanded capabilities for optical stimulation as well as simultaneous electrical neural recordings, as with an optrode array. Temporal response of light outputs and light stability during commutator rotation were assessed. The influence of high current optical control signals on adjacent neural recording channels was also explored. To validate the function of this LED based system in in vivo recording scenarios, chronic stimulation experiments were performed.

Significant progress has been made in the application of optogenetic stimulation as a means to modulate and control cellular functions within chemically-identical groups of cells. High resolution imaging can detect subtle morphological (shape/refractive index) changes in cells subsequent to optogenetic stimulation. Invasive topographical measurement methods such as mainstream AFM and other scanning probe techniques suffer from low temporal resolution and restricted field of view, resulting in reduced throughput, even though these methods exhibit high sensitivity to morphological changes. QPM, integrated with optogenetic stimulation incorporates a wide-field, label-free, non-invasive optical imaging technique for all optical stimulation and detection with high spatial and temporal resolution. We dynamically monitored phase of cells, sensitized with and without ChR2, using quantitative phase microscopy with and without light stimulation. The variation of phase in optogenetically stimulated cells (expressing ChR2) was found to be higher than that of the control cells. We report that our method could potentially evaluate effectiveness of various opsins and stimulation parameters including cellular function under different physiological surroundings via spatiallymodulated optogenetic stimulation and wide-field quantitative phase imaging.