Introduction: Unilateral Carotid Occlusion (UCO) serves as a model of partial cerebral ischemia which mimics clinical situations such as stenosis or atherosclerosis. UCO has known to have slight and merely short-term effects on cerebral hemodynamic and metabolic functions. The aim of this study was to test the effects of UCO compared to bilateral carotid occlusion (BCO) on the responses of the brain to spreading depression (SD). Methods: Rats were monitored up to 24 hours after UCO and BCO using a Multi-Site - Multi-Parametric (MSMP) system, which evaluates mitochondrial function using the NADH fluorometry and CBF using laser Doppler flowmetry. The induction of SD and the exposure to short anoxia served as tools to investigate the effects of UCO and BCO on the brain. Results: UCO and BCO led to a short lasting decrease in CBF and an increase in NADH. During SD waves and short anoxia a hyperemic response occurred, which decreased 24 hours following UCO in the hemisphere ipsilateral to the occluded artery and increased in the contralateral hemisphere. The hyperemic response decreased in both hemispheres 24 hours following BCO. NADH levels during SD waves increased in the hemisphere ipsilateral to the occluded artery following UCO and in both hemispheres following BCO, but remained similar to control levels during short anoxia. Conclusions: UCO leads to long term alterations in cerebral blood supply, which may be detected 24 hours following such occlusion. These changes are minor compared to the effect of BCO and have minimal influence on mitochondrial function.

In this study, we explore the hemodynamic response in the lesioned rat spinal cord following peripheral nerve stimulation. Oxy and deoxy hemoglobin were measured (using a four color LED multispectral intrinsic optical imaging system) simultaneously with blood flow (laser speckle measurement). Both optical and electrophysiological data are compared spatially and against stimulation strength. When compared with non-lesioned animals, the hemodynamic response is seen to display significant differences exhibiting increased initial dip and decreased blood drain following stimulation. The origin of the difference is observed to be due to the vascular nature of the injury. The distinct hemodynamic responses may have a strong impact on General Linear Model based fMRI studies of spinal cord lesions due to the difficulty in separating vascular effects from neuronal plasticity following injury.

Cerebral palsy (CP) is the most common motor disorder of central origin in childhood and affects at least 2 children per 1000 live births every year. Neuroimaging techniques are needed to study neuroplastic rearrangements in the human brain in vivo as a result of CP. Unfortunately, accurate imaging from currently available techniques often requires the patients' complete body confinement, steadiness and minimal noise for a long period of time, which limits the success rate to less than 50% for normal children and worse for CP-affected ones. In this work we show that functional near infrared (fNIR) imaging is robust to motion artifacts and has excellent potential as a sensitive diagnostic tool for this motor disorder. We have analyzed data from pediatric normal and CP patients performing finger-tapping and handwaving motor cortex activation tasks. From these analyses we have identified both spatial and temporal metrics of NIR-based motor cortex activation patterns that can clearly distinguish between normal and CP patients. We also present data from additional patients where signal processing methods are applied to filter out concurrently recorded hemodynamic signals due to breathing and cardiac pulsation. It is shown that filtering can substantially improve the quality of activation data, thus enabling more accurate comparison of activation patterns between normal and CP-affected children.

Voltage sensitive fluorescent dyes have long been used to measure physiological voltages in live cell cultures. However dyes suffer from poor contrast and limited recording duration due to photobleaching. A photostable voltage sensitive cellular label, such as a noble metal nanoparticle, would potentially allow for indefinite recording from neural and other live cell cultures. Noble metals possess an inherent voltage sensitivity: their optical properties depend on their density of free electrons, which can be modulated in an aqueous environment by charging or discharging the double layer capacitance with an applied voltage. This manuscript contains a simple analysis of the expected voltage sensitivity using gold nanospheres and nanoshells in both darkfield and photothermal detection modalities and concludes that high bandwidth voltage measurement is fundamentally achievable.

Spectral Domain Phase Microscopy (SDPM) is a recent extension of Spectral Domain Optical Coherence Tomography (SDOCT) that exploits the extraordinary phase stability of spectrometer-based systems with common-path geometry to resolve sub-wavelength displacements within a sample volume. This technique has been implemented for high resolution axial displacement and velocity measurements in biological samples, but since axial displacement information is acquired serially, has been unable to measure fast temporal dynamics in extended samples. Depth-Encoded SDPM (DESDPM) uses multiple sample arms with unevenly spaced common path reference reflectors to multiplex independent SDPM signals from separate lateral positions on a sample simultaneously using a single interferometer, thus limiting the time required to detect unique optical events to the integration time of the detector. The minimum measured sample displacements determined from the standard deviation of the detected phase as a function of time two ideal reflectors were 407 and 730 pm. Heat-induced expansion in a microscope slide was measured at two sites simultaneously. A 51 ms delay in 50% rise time of the surface displacement was measured. Further application of this technique to biological samples could yield insight into temporal dynamics of activation signals.

Flavoprotein autofluorescence is an activity dependent intrinsic signal. Flavoproteins are involved in the electron transport chain and change their fluorescence according to the cellular redox state. We have been using flavoprotein autofluorescence in the cerebellum to examine properties of cerebellar circuits. Studies have also focused on understanding the cellular and metabolic origins of this intrinsic optical signal. Parallel fiber stimulation evokes a beamlike response intersected by bands of decreased fluorescence. The beam response is biphasic, with an early fluorescence increase (light phase) followed by a slower decrease (dark phase). We show this signal originates from flavoproteins as determined by its wavelength selectivity and sensitivity to blockers of the electron transport chain. Selectively blocking glutamate receptors abolished the on-beam light phase with the dark phase remaining intact. This demonstrates that the light phase is due to postsynaptic neuronal activation and suggests the dark phase is primarily due to glial activation. The bands of reduced fluorescence intersecting the beam are primarily neuronal in origin, mediated by GABAergic transmission, and due to the inhibitory action of molecular layer interneurons on Purkinje cells and the interneurons themselves. This parasagittally organized molecular layer inhibition differentially modulates the spatial pattern of cerebellar cortical activity. Flavoprotein imaging also reveals the functional architectures underlying the responses to inferior olive and peripheral whisker pad stimulation. Therefore, flavoprotein autofluorescence imaging is providing new insights into cerebellar cortical function and neurometabolic coupling.

Flavoprotein autofluorescence optical imaging is developing into a powerful research tool to study neural activity, particularly in vivo. In this study we used this imaging technique to investigate the neuronal mechanism underlying the episodic movement disorder that is characteristic of the tottering (tg) mouse, a model of episodic ataxia type 2. Both EA2 and the tg mouse are caused by mutations in the gene encoding Ca_v2.1 (P/Q-type) voltage-gated Ca²⁺ channels. These mutations result in a reduction in P/Q Ca²⁺ channel function. Both EA2 patients and tg mice have a characteristic phenotype consisting of transient motor attacks triggered by stress, caffeine or ethanol. The neural events underlying these episodes of dystonia are unknown. Flavoprotein autofluorescence optical imaging revealed spontaneous, transient, low frequency oscillations in the cerebellar cortex of the tg mouse. Lasting from 30 - 120 minutes, the oscillations originate in one area then spread to surrounding regions over 30 - 60 minutes. The oscillations are reduced by removing extracellular Ca²⁺ and blocking Ca_v 1.2/1.3 (L-type) Ca²⁺ channels. The oscillations are not affected by blocking AMPA receptors or by electrical stimulation of the parallel fiber - Purkinje cell circuit, suggesting the oscillations are generated intrinsically in the cerebellar cortex. Conversely, L-type Ca²⁺ agonists generate oscillations with similar properties. In the awake tg mouse, transcranial flavoprotein imaging revealed low frequency oscillations that are accentuated during caffeine induced attacks of dystonia. The oscillations increase during the attacks of dystonia and are coupled to oscillations in face and hindlimb EMG activity. These transient oscillations and the associated cerebellar dysfunction provide a novel mechanism by which an ion channel disorder results in episodic motor dysfunction.

The mechanisms of learning and memory are the most important functions in an animal brain. Investigating neuron circuits and network maps in a brain is the first step toward understanding memory and learning behavior. Since Drosophila brain is the major model for understanding brain functions, we measure the florescence lifetimes of different GFP-based reporters expressed in a fly brain. In this work, two Gal4 drivers, OK 107 and MZ 19 were used. Intracellular calcium ([Ca²⁺]) concentration is an importation indicator of neuronal activity. Therefore, several groups have developed GFP-based calcium sensors, among which G-CaMP is the most popular and reliable. The fluorescence intensity of G-CaMP will increase when it binds to calcium ion; however, individual variation from different animals prevents quantitative research. In this work, we found that the florescence lifetime of G-CaMP will shrink from 1.8 ns to 1.0 ns when binding to Ca²⁺. This finding can potentially help us to understand the neuron circuits by fluorescence lifetime imaging microscopy (FLIM). Channelrhodopsin-2 (ChR2) is a light-activated ion-channel protein on a neuron cell membrane. In this work, we express ChR2 and G-CaMP in a fly brain. Using a pulsed 470-nm laser to activate the neurons, we can also record the fluorescence lifetime changes in the structure. Hence, we can trace and manipulate a specific circuit in this animal. This method provides more flexibility in brain research.

This paper highlights how the genetic incorporation of artificial opsins into the retina can lead to a new class of retinal prosthesis. We demonstrate the efficacy of incorporating channelrhodopsin into neuron cells in-vitro and show how that can be scaled to in-vivo. We show that we need typically 100mW/cm² of instantaneous light intensity on the neuron in order to stimulate action potentials which results in 10W/cm² required from the light source. We thus use GaN LED arrays to provide spatially controlled stimulation which is of sufficient brightness to stimulate the cells.

A novel, spatially selective method to stimulate cranial nerves has been proposed: contact free stimulation with optical radiation. The radiation source is an infrared pulsed laser. The Case Report is the first report ever that shows that optical stimulation of the auditory nerve is possible in the human. The ethical approach to conduct any measurements or tests in humans requires efficacy and safety studies in animals, which have been conducted in gerbils. This report represents the first step in a translational research project to initiate a paradigm shift in neural interfaces. A patient was selected who required surgical removal of a large meningioma angiomatum WHO I by a planned transcochlear approach. Prior to cochlear ablation by drilling and subsequent tumor resection, the cochlear nerve was stimulated with a pulsed infrared laser at low radiation energies. Stimulation with optical radiation evoked compound action potentials from the human auditory nerve. Stimulation of the auditory nerve with infrared laser pulses is possible in the human inner ear. The finding is an important step for translating results from animal experiments to human and furthers the development of a novel interface that uses optical radiation to stimulate neurons. Additional measurements are required to optimize the stimulation parameters.

Optical stimulation (OS) is a relatively novel approach for restoring function to the damaged nervous system. The effectiveness and safety of OS is dependent upon selecting the appropriate stimulation parameters. This involves stimulating neurons to their activation threshold while preventing laser-induced tissue injury. Although significant advances have been made in studying the efficacy of OS, safety parameters are still being developed. We have employed electrophysiological techniques to determine salient experimental parameters of safety that can be used to optimize OS. Extracellular recordings of compound nerve potentials were obtained from excised adult rat sciatic nerves. OS was accomplished with infrared pulsed Nd:YAG, Er:YAG and diode lasers that had peak wavelength emissions at 1.064 μm, 2.94 μm and 1.85 μm, respectively. Electrically evoked compound action potentials (E-CAPs) were assayed before and after laser irradiation to determine if OS affected E-CAPs. Injurious laser irradiation doses were observed at levels 2-3 fold greater than optical threshold, producing tissue hyalinization and decreases in the peak amplitude of E-CAPs. However, effects on electrical threshold and conduction velocity were negligible. At laser irradiation doses near optical threshold, low repetition rates of laser pulses produced a gradual increase in laser evoked CAP (L-CAP) amplitudes, suggesting a cumulative effect in the interactions between light and tissue. Higher repetition rates (5-10 Hz) at laser irradiation doses 2-3 fold above optical threshold produced a decrement in L-CAP and E-CAP amplitudes. These results suggest that laser pulse parameters have a direct impact on optical stimulation and damage thresholds.

Infrared Nerve Stimulation (INS) offers several advantages over electrical stimulation, including more precise spatial selectivity and improved surgical access. In this study, INS and electrical stimulation were compared in their ability to activate the vestibular branch of the VIII^th nerve, as a potential way to treat balance disorders. The superior and lateral canals of the vestibular system of Guinea pigs were identified and approached with the aid of precise 3-D reconstructions. A monopolar platinum stimulating electrode was positioned near the ampullae of the canals, and biphasic current pulses were used to stimulate vestibular evoked potentials and eye movements. Thresholds and input/output functions were measured for various stimulus conditions. A short pulsed diode laser (Capella, Lockheed Martin-Aculight, Inc., Bothell WA) was placed in the same anatomical position and various stimulus conditions were evaluated in their ability to evoke similar potentials and eye movements.

Lasers can be used to stimulate neural tissue, including the sciatic nerve or auditory neurons. Wells and coworkers suggested that neural tissue is likely stimulated by heat.[1,2] Ion channels that can be activated by heat are the TRPV channels, a subfamily of the Transient Receptor Potential (TRP) ion channels. TRPV channels are nonselective cation channels found in sensory neurons involved in nociception. In addition to various chemicals, TRPV channels can also be thermally stimulated. The activation temperature for the different TRPV channels varies and is 43°C for TRPV1 and 39°C for TRPV3. By performing an immunohistochemical staining procedure on frozen 20 μm cochlear slices using a primary TRPV1 antibody, we observed specific immunostaining of the spiral ganglion cells. Here we show that in mice that lack the gene for the TRPV1 channel optical radiation cannot evoke action potentials on the auditory nerve.

In the 1990's a new laser technology, Vertical Cavity Surface Emitting Lasers, or VCSELs, emerged and transformed the data communication industry. The combination of performance characteristics, reliability and performance/cost ratio allowed high data rate communication to occur over short distances at a commercially viable price. VCSELs have not been widely used outside of this application space, but with the development of new attributes, such as a wider range of available wavelengths, the demonstration of arrays of VCSELs on a single chip, and a variety of package form factors, VCSELs can have a significant impact on medical diagnostic and therapeutic applications. One area of potential application is neurostimulation. Researchers have previously demonstrated the feasibility of using 1850nm light for nerve stimulation. The ability to create an array of VCSELs emitting at this wavelength would allow significantly improved spatial resolution, and multiple parallel channels of stimulation. For instance, 2D arrays of 100 lasers or more can be integrated on a single chip less than 2mm on a side. A second area of interest is non-invasive sensing. Performance attributes such as the narrow spectral width, low power consumption, and packaging flexibility open up new possibilities in non-invasive and/or continuous sensing. This paper will suggest ways in which VCSELs can be implemented within these application areas, and the advantages provided by the unique performance characteristics of the VCSEL. The status of VCSEL technology as a function of available wavelength and array size and form factors will be summarized.

Cellular exposure to nanosecond duration electrical pulses (nsEP) has been shown to elicit a marked decrease in plasma membrane resistance hypothesized to be due to formation of nanopores. Patch clamp technique has been used to measure changes in plasma membrane resistance immediately following nsEP exposure. Previous research by our group identified absorbed dose as a single metric to quantify cellular exposure across various pulse parameters. In that work, the relationship between the drop in membrane resistance and absorbed dose was hypothesized to be a measure of cell sensitivity to nsEP. In this study, we explore this hypothesis by employing patch clamp technique prior to exposure to measure whole cell currents at time points before and after exposure. Using two cell lines (GH3 and CHO), dose response curves were generated for single 60ns pulse exposures at 10 and 90 seconds post exposure. From this data, it was evident that dose response curves at 10 seconds post exposure showed increased permeability following nsEP exposure compared to 90 seconds post exposure. We attribute this difference to active recovery of the cell to nanoporation. However, at 90 seconds, the results for both cell types appeared to nearly match previously published results proving that a cell in whole cell configuration is slightly more sensitive to nsEP exposure. Future work will extend this study to include multiple nsEP exposures and additional cell lines.

It has been one of the most discussed and intriguing topics -the quest to control neural circuitry as a precursor to decoding the operations of the human brain and manipulating its diseased state. Electrophysiology has created a gateway to control this circuitry with high precision. However, it is not practical to apply these techniques to living systems because these techniques are invasive and lack the spatial resolution necessary to properly address various neural cell components, cell assemblies or even tissues. Here we describe a new instrument that has the potential to replace the conventional patch clamping technique, the workhorse of neural physiology. A Digital Light Processing system from Texas Instruments and an Olympus IX71 inverted microscope were combined to achieve neuronal control at a subcellular spatial resolution. Accompanying these two technologies can be almost any light source, and for these experiments a pair of pulsed light sources that produced two pulse trains at different wavelengths tuned to activate or inactivate selectively the ChR2 and NpHR channels that were cloned to express light sensitive versions in neurons. Fura- 2 ratiometric fluorescent dye would be used to read-out calcium activity. The Pulsed light sources and a filter wheel are under computer control using a National Instruments digital control board and a CCD camera used to acquire real time cellular responses to the spatially controlled pulsed light channel activation would be controlled and synchronized using NI LabVIEW software. This will provide for a millisecond precision temporal control of neural circuitry. Thus this technology could provide researchers with an optical tool to control the neural circuitry both spatially and temporally with high precision.