Cross-polarized reflected light measurement of fast optical responses associated with neural activation

We developed an optical probe for cross-polarized reflected light measurements and investigated optical signals associated with electrophysiological activation in isolated lobster nerves. The cross-polarized baseline light intensity (structural signal) and the amplitude of the transient response to stimulation (functional signal) measured in reflected mode were dependent on the orientation of the nerve axis relative to the polarization plane of incident light. The maximum structural signal and functional response amplitude were observed at 45 degrees , and the ratio of functional to structural signals was approximately constant across orientations. Functional responses were measured in single trials in both transmitted and reflected geometries and responses had similar waveforms. Both structural and functional signals were an order of magnitude smaller in reflected than in transmitted light measurements, but functional responses had similar signal/noise ratios. We propose a theoretical model based on geometrical optics that is consistent with experimental results. In the model, the cross-polarized structural signal results from light reflection from axonal fibers and the transient functional response arises from axonal swelling associated with neural activation. Polarization-sensitive reflected light measurements could greatly enhance in vivo imaging of neural activation since cross-polarized responses are much larger than scattering signals now employed for dynamic functional neuroimaging.     

Whole Cell Patch Clamp for Investigating the Mechanisms of Infrared Neural Stimulation

It has been demonstrated in recent years that pulsed, infrared laser light can be used to elicit electrical responses in neural tissue, independent of any further modification of the target tissue. Infrared neural stimulation has been reported in a variety of peripheral and sensory neural tissue in vivo, with particular interest shown in stimulation of neurons in the auditory nerve. However, while INS has been shown to work in these settings, the mechanism (or mechanisms) by which infrared light causes neural excitation is currently not well understood. The protocol presented here describes a whole cell patch clamp method designed to facilitate the investigation of infrared neural stimulation in cultured primary auditory neurons. By thoroughly characterizing the response of these cells to infrared laser illumination in vitro under controlled conditions, it may be possible to gain an improved understanding of the fundamental physical and biochemical processes underlying infrared neural stimulation.     

Vagus nerve bundle stimulation using 1505-nm laser irradiation in an in-vivo rat model

Laser nerve stimulation using near-infrared laser irradiation has recently been studied in the peripheral nervous system as an alternative method to conventional electrical nerve stimulation. Bringing this method to the vagus nerve model could leverage this emerging stimulation approach to be tested in broader preclinical applications. Here, we report the capability of the laser nerve stimulation method on the rat vagus nerve bundle with a 1505-nm diode laser operated in continuous-wave mode. Studies of the stimulation threshold and laser-induced acute thermal injury to the nerve bundle were also performed to determine a temperature window for safe, reliable and reproducible laser stimulation of the rat vagus nerve bundle. The results show that laser stimulation of the vagus nerve bundle provides reliable and reproducible nerve stimulation in a rat model. These results also confirm a threshold temperature of >42°C with acute nerve damage observed above 46°C. A strong correlation was obtained between the laser time required to raise the nerve temperature above the stimulation threshold and the mean arterial pressure response. Advantages of the method such as non-contact delivery of external stimulus signals at mm scaled distance in air, enhanced spatial selectivity and electrical artefact-free measurements may indicate its potential to counteract the side effects of conventional electrical vagus nerve stimulation.     

Axonal model for temperature stimulation

Recent studies indicate that a rapid increase in local temperature plays an important role in nerve stimulation by laser. To analyze the temperature effect, our study modified the classical HH axonal model by incorporating a membrane capacitance-temperature relationship. The modified model successfully simulated the generation and propagation of action potentials induced by a rapid increase in local temperature when the Curie temperature of membrane capacitance is below 40 °C, while the classical model failed to simulate the axonal excitation by temperature stimulation. The new model predicts that a rapid increase in local temperature produces a rapid increase in membrane capacitance, which causes an inward membrane current across the membrane capacitor strong enough to depolarize the membrane and generate an action potential. If the Curie temperature of membrane capacitance is 31 °C, a temperature increase of 6.6–11.2 °C within 0.1–2.6 ms is required for axonal excitation and the required increase is smaller for a faster increase. The model also predicts that: (1) the temperature increase could be smaller if the global axon temperature is higher; (2) axons of small diameter require a smaller temperature increase than axons of large diameter. Our study indicates that the axonal membrane capacitance-temperature relationship plays a critical role in inducing the transient membrane depolarization by a rapidly increasing temperature, while the effects of temperature on ion channel kinetics cannot induce depolarization. The axonal model developed in this study will be very useful for analyzing the axonal response to local heating induced by pulsed infrared laser.     

Influence of frequency and temperature on the mechanisms of nerve conduction block induced by high-frequency biphasic electrical current

The influences of stimulation frequency and temperature on mechanisms of nerve conduction block induced by high-frequency biphasic electrical current were investigated using a lumped circuit model of the myelinated axon based on Schwarz and Eikhof (SE) equations. The simulation analysis showed that a temperature-frequency relationship was determined by the axonal membrane dynamics (i.e. how fast the ion channels can open or close.). At a certain temperature, the axonal conduction block always occurred when the period of biphasic stimulation was smaller than the action potential duration (APD). When the temperature decreased from 37 to 15 degrees C, the membrane dynamics slowed down resulting in an APD increase from 0.4 to 2.4 ms accompanied by a decrease in the minimal blocking frequency from 4 to 0.5 kHz. The simulation results also indicated that as the stimulation frequency increased the mechanism of conduction block changed from a cathodal/anodal block to a block dependent upon continuous activation of potassium channels. Understanding the interaction between the minimal blocking frequency and temperature could promote a better understanding of the mechanisms of high frequency induced axonal conduction block and the clinical application of this method for blocking nerve conduction.     

Effects of Q-switched Nd:YAG laser irradiation on neural impulse propagation: II. Dorsal roots and peripheral nerves.

We have studied the effects of Q-switched Nd:YAG laser irradiation on transmission of neural impulses in sensory nerve fibers in anesthetized rats and cats. Laser light was applied to dorsal roots (rat, cat) and to the sciatic nerve (rat) at increasing pulse energies ranging from 10 to 100 mJ/pulse for 5 minutes each. Compound action potentials recorded from dorsal roots and the sciatic nerve in response to high intensity electrical stimulation during laser application at increasing pulse energies showed a progressive preferential reduction of the slow late component of the electrically evoked response. Preliminary data from multifilament recordings from dorsal roots in cats demonstrated that conduction in small slow conducting fibers was blocked at lower laser pulse energies than in fibers with faster conduction velocities. These results imply, that laser light might have differential effects on slow versus fast conducting sensory nerve fibers. It is most likely that the preferential effect of laser irradiation on slow conducting fibers is mediated by photothermal mechanisms, since temperature increased substantially during laser application.     

Precise spatiotemporal control of optogenetic activation using an acousto-optic device

Light activation and inactivation of neurons by optogenetic techniques has emerged as an important tool for studying neural circuit function. To achieve a high resolution, new methods are being developed to selectively manipulate the activity of individual neurons. Here, we report that the combination of an acousto-optic device (AOD) and single-photon laser was used to achieve rapid and precise spatiotemporal control of light stimulation at multiple points in a neural circuit with millisecond time resolution. The performance of this system in activating ChIEF expressed on HEK 293 cells as well as cultured neurons was first evaluated, and the laser stimulation patterns were optimized. Next, the spatiotemporally selective manipulation of multiple neurons was achieved in a precise manner. Finally, we demonstrated the versatility of this high-resolution method in dissecting neural circuits both in the mouse cortical slice and the Drosophila brain in vivo. Taken together, our results show that the combination of AOD-assisted laser stimulation and optogenetic tools provides a flexible solution for manipulating neuronal activity at high efficiency and with high temporal precision.     

Gold Nanorod-assisted Optical Stimulation of Neuronal Cells

Recent studies have demonstrated that nerves can be stimulated in a variety of ways by the transient heating associated with the absorption of infrared light by water in neuronal tissue. This technique holds great potential for replacing or complementing standard stimulation techniques, due to the potential for increased localization of the stimulus and minimization of mechanical contact with the tissue. However, optical approaches are limited by the inability of visible light to penetrate deep into tissues. Moreover, thermal modelling suggests that cumulative heating effects might be potentially hazardous when multiple stimulus sites or high laser repetition rates are used. The protocol outlined below describes an enhanced approach to the infrared stimulation of neuronal cells. The underlying mechanism is based on the transient heating associated with the optical absorption of gold nanorods, which can cause triggering of neuronal cell differentiation and increased levels of intracellular calcium activity. These results demonstrate that nanoparticle absorbers can enhance and/or replace the process of infrared neural stimulation based on water absorption, with potential for future applications in neural prostheses and cell therapies.     

Neural driven angiogenesis by overexpression of nerve growth factor

Mechanisms regulating angiogenesis are crucial in adjusting tissue perfusion on metabolic demands. We demonstrate that overexpression of nerve growth factor (NGF) in brown adipose tissue (BAT) of NGF-transgenic mice elevates both mRNA and protein levels of vascular endothelial growth factor (VEGF) and VEGF-receptors. Increased vascular permeability, leukocyte–endothelial interactions (LEI), and tissue perfusion were measured using intravital microscopy. NGF-stimulation of adipocytes and endothelial cells elevates mRNA expression of VEGF and its receptors, an effect blocked by NGF neutralizing antibodies. These data suggest an activation of angiogenesis as a result of both: stimulation of adipozytes and direct mitogenic effects on endothelial cells. The increased nerve density associated with vessels strengthened our hypothesis that tissue perfusion is regulated by neural control of vessels and that the interaction between the NGF and VEGF systems is the critical driver for the activated angiogenic process. The interaction of VEGF- and NGF-systems gives new insights into neural control of organ vascularization and perfusion.     

Induction of neuronal activation by femtosecond‐pulsed laser irradiation and its potential application for amyloid‐β–induced toxicity assessment

Manipulating neural activity is crucial for studying the neural connectivity and the pathophysiology of neurodegenerative disease. Among various techniques for neural activation, direct optical stimulation method with femtosecond‐pulsed laser is simple and can be specifically applied on a single neuron. Brief irradiation of femtosecond laser pulses on a neuron elevates intracellular calcium, and it propagates to adjacent neurons. However, the mechanisms of laser‐induced neural activation are still unclear. In this report, we have elucidated the mechanism of laser‐induced neural activation which could be mediated by superoxide, specifically blocked by diphenyleneiodonium chloride, and depletion in intracellular calcium storage. Furthermore, we also showed that the propagation of calcium initiated by laser stimulation is dependent on the presence of extracellular calcium as well as electrical and chemical synapses. We verified the applicability of such mechanism for the assessment of neuronal functionality, by measuring calcium elevation, intracellular calcium propagation, ROS increase, and performing cell death assay in vehicle and Aβ‐treated neurons. This work suggests promising applications of the potential for implementing such laser‐induced neural activation for rapid and reliable drug screening.

     

Investigation of the mechanisms for wireless nerve stimulation without active electrodes

Electric-field stimulation of neuronal activity can be used to improve the speed of regeneration for severed and damaged nerves. Most techniques, however, require invasive electronic circuitry which can be uncomfortable for the patient and can damage surrounding tissue. A recently suggested technique uses a graft-antenna—a metal ring wrapped around the damaged nerve—powered by an external magnetic stimulation device. This technique requires no electrodes and internal circuitry with leads across the skin boundary or internal power, since all power is provided wirelessly. This paper examines the microscopic basic mechanisms that allow the magnetic stimulation device to cause neural activation via the graft-antenna. A computational model of the system was created and used to find that under magnetic stimulation, diverging electric fields appear at the metal ring's edges. If the magnetic stimulation is sufficient, the gradients of these fields can trigger neural activation in the nerve. In-vivo measurements were also performed on rat sciatic nerves to support the modeling finding that direct contact between the antenna and the nerve ensures neural activation given sufficient magnetic stimulation. Simulations also showed that the presence of a thin gap between the graft-antenna and the nerve does not preclude neural activation but does reduce its efficacy.     

Light-addressed single-neuron stimulation in dissociated neuronal cultures with sparse expression of ChR2

Individual neurons are heterogeneous and have profound impact on population activity in a complex cortical network. Precise experimental control of the firing of multiple neurons would be therefore beneficial to advance our understanding of cell-network interactions. Except for direct intracellular stimulation, however, it is difficult to gain precise control of targeted neurons without inducing antidromic activation of untargeted neurons. To overcome this problem, we attempt to create a sparse group of photosensitized neurons via transfection of Channelrhodopsin-2 (ChR2) in primary dissociated cultures and then deliver light-addressed stimulation exclusively to these target neurons. We first show that liposome transfection was able to express ChR2 in 0.3-1.9% of cells plated depending on cell density. This spatially sparse but robust expression in our neuronal cultures offered the capability of single cell activation by illuminating a spot of light. We then demonstrated that delivering a pulsed train to photo-activate a single neuron had a substantial effect on the activity level of an entire neuronal culture. Furthermore, the activity level was controllable by altering the frequency of light illumination when 4 neurons were recruited as stimulation targets. These results suggest that organized activation of a very small population of neurons can provide better control over global activity of neuronal circuits than can single-neuron activities by themselves.     

Signalling mechanisms in the regulation of vacuolar ion release in guard cells

Pharmacological agents were used to investigate the possible involvement of actin in signalling chains associated with abscisic acid (ABA)-induced ion release from the guard cell vacuole, a process which is absolutely essential for stomatal closure. Effects on the ABA-induced transient stimulation of tonoplast efflux were measured, using (86)Rb in isolated guard cells of Commelina communis, together with effects on stomatal apertures. In the response to 10 microm ABA (triggered by Ca(2+) influx rather than internal Ca(2+) release), jasplakinolide (stabilizing actin filaments) and latrunculin B (depolymerizing actin filaments) had opposite effects. Both closure and the vacuolar efflux transient were inhibited by jasplakinolide but enhanced by latrunculin B. At 10 microm ABA prevention of mitogen-activated protein (MAP) kinase activation by PD98059 partially inhibited closure and reduced the efflux transient. By contrast, latrunculin B inhibited the efflux transient at 0.1 microm ABA (involving internal Ca(2+) release rather than Ca(2+) influx). The results suggest that 10 microm ABA activates Ca(2+)-dependent vacuolar ion efflux via a Ca(2+)-permeable influx channel which is maintained closed by interaction with F-actin. A MAP kinase is also involved, in a chain similar to that postulated for Ca(2+)-dependent gene expression in cold acclimation.     

Membrane fusion machines of paramyxoviruses: capture of intermediates of fusion

Peptides derived from heptad repeat regions adjacent to the fusion peptide and transmembrane domains of many viral fusion proteins form stable helical bundles and inhibit fusion specifically. Paramyxovirus SV5 fusion (F) protein-mediated fusion and its inhibition by the peptides N-1 and C-1 were analyzed. The temperature dependence of fusion by F suggests that thermal energy, destabilizing proline residues and receptor binding by the hemagglutinin-neuraminidase (HN) protein collectively contribute to F activation from a metastable native state. F-mediated fusion was reversibly arrested by low temperature or membrane-incorporated lipids, and the resulting F intermediates were characterized. N-1 inhibited an earlier F intermediate than C-1. Co-expression of HN with F lowered the temperature required to attain the N-1-inhibited intermediate, consistent with HN binding to its receptor stimulating a conformational change in F. C-1 bound and inhibited an intermediate of F that could be detected until a point directly preceding membrane merger. The data are consistent with C-1 binding a pre-hairpin intermediate of F and with helical bundle formation being coupled directly to membrane fusion.     

滨海电厂冷却水余热和余氯对中华哲水蚤的影响

依据滨海电厂冷却系统的实际运作情况,模拟研究了滨海电厂冷却水余热和余氯对中华哲水蚤的影响.结果表明：中华哲水蚤的热忍受能力随暴露时间延长而降低,随驯化温度升高而升高.驯化温度为16 ℃～27 ℃的中华哲水蚤在持续受到15、30、45 min热冲击及持续升温暴露24和48 h的致死温度分别为29.9 ℃～31.7 ℃、29.4 ℃～31.0 ℃、28.9 ℃～30.3 ℃和26.9 ℃～28.5 ℃、26.4 ℃～28.0 ℃.当驯化温度升高到一定程度后,其热忍受能力不再上升.电厂冷却水中余氯对中华哲水蚤的毒性随驯化温度升高、升温幅度增大及暴露时间延长而增强.     

Turnover of rod photoreceptor outer segments. I. Membrane addition and loss in relationship to temperature

Membrane turnover in outer segments of Rana pipiens red rods (ROS) was studied in tadpoles maintained under cyclic lighting (12L:12D) at 23 degrees, 28 degrees, and 33 degrees C. Large fragments (greater than 2 microns in diameter or length) were shed from the ROS tips shortly after the onset of light. These were phagocytized by the pigment epithelium (PE) which caused an increase in the number of phagosomes greater than 2 microns in size (large phagosomes). Large phagosomes were present in highest numbers 2-4 h after light exposure and were degraded by 8-12 h. The proportion of ROS that shed each day after the onset of the light cycle increased with increment increases in temperatures (23 degrees C-18%, 28 degrees C-33%, 33 degrees C-42% per day), resulting, in a reduction in the average interval of time between repeated sheddings (23 degrees C-5.6 days, 28 degrees C-3 days, 33 degrees C-2.4 days) though the average numbers of disks shed from ROS at the various temperatures were not significantly different (23 degrees C-139.5 +/- 5.7, 28 degrees C-129.4 +/- 7.6, 33 degrees C-129.9 +/- 4.8 disks/shed packet). Phagosomes in the PE that were less than 2 microns in diameter (small phagosomes) were present in relatively constant numbers throughout the day, and their numbers increased at higher temperatures. The absence of a concomitant increase in small phagosomes as large phagosomes were degraded indicates that large phagosomes were not the major source of small phagosomes. When the PE was isolated to culture in the absence of the retina, these small phagosomes were degraded. The rate of disk addition to the ROS base was determined by autoradiography after [3H]leucine injection. The number of disks added per day increased with elevations of temperature (23 degrees C-32.4; 28 degrees C-55.9; 33 degrees C-65.5). The average number of disks added to the ROS between repeated sheddings (23 degrees C-181.4; 28 degrees C-167.7; 33 degrees C-157.2) was greater than the number of disks shed after light exposure. Inasmuch as the ROS show no net increase in length during the tadpole stages utilized, the remaining disks must be lost at some other time. Electron microscope analysis revealed the presence of small groups of disks in curled configurations at the tips of ROS, suggesting possible stages of detachment.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vivo evidence for short- and long-range cell communication in cranial neural crest cells

The proper assembly of craniofacial structures and the peripheral nervous system requires neural crest cells to emerge from the neural tube and navigate over long distances to the branchial arches. Cell and molecular studies have shed light on potential intrinsic and extrinsic cues, which, in combination, are thought to ensure the induction and specification of cranial neural crest cells. However, much less is known about how migrating neural crest cells interpret and integrate signals from the microenvironment and other neural crest cells to sort into and maintain the stereotypical pattern of three spatially segregated streams. Here, we explore the extent to which cranial neural crest cells use cell-to-cell and cell-environment interactions to pathfind. The cell membrane and cytoskeletal elements in chick premigratory neural crest cells were labeled in vivo. Three-dimensional reconstructions of migrating neural crest cells were then obtained using confocal static and time-lapse imaging. It was found that neural crest cells maintained nearly constant contact with other migrating neural crest cells, in addition to the microenvironment. Cells used lamellipodia or short, thin filopodia (1-2 microm wide) for local contacts (<20 microm). Non-local, long distance contact (up to 100 microm) was initiated by filopodia that extended and retracted, extended and tracked, or tethered two non-neighboring cells. Intriguingly, the cell-to-cell contacts often stimulated a cell to change direction in favor of a neighboring cell's trajectory. In summary, our results present in vivo evidence for local and long-range neural crest cell interactions, suggesting a possible role for these contacts in directional guidance.     

Succinoylation of sugarcane bagasse under ultrasound irradiation

The chemical modification of sugarcane bagasse with succinic anhydride using pyridine as solvent after ultrasound irradiation was studied. The optimized parameters included ultrasound irradiating time 0-50 min, reaction time 30-120 min, succinic anhydride concentration by the ratio of dried sugarcane bagasse to succinic anhydride from 1:0.25 to 1:1.50, and reaction temperature 75-115 degrees C are required in the process. The extent of succinoylation was measured by the weight percent gain (WPG), which increased with increments of reaction time, succinic anhydride concentration, and reaction temperature. The ultrasound irradiation has a positive effect on bagasse succinoylation process. On the other hand, the ultrasonic pre-treatment application broke down the cell wall polymers, resulting in, therefore, a negative effect on the WPG. Evidences of succinoylation were also provided by FT-IR and CP MAS (13)C NMR and the results showed that the succinoylation at C-2 and C-3 occurred. The thermal stability of the succinylated bagasse decreased upon chemical modification.     

Multiphoton stimulation of neurons

We pulsed the activation of neurons using a femtosecond laser. Pyramidal neurons are depolarized and fire action potentials when high intensity mode-locked infrared light irradiates somatic membranes and axon initial segments. This depolarization is reversible, does not occur with CW laser light, and appears to be due to multiphoton excitation. We describe two regimes of multiphoton optical stimulation. Low intensity, long duration laser irradiation produces a sustained depolarization, insensitive to sodium channel blockers yet sensitive to antioxidants. On the other hand, high intensity, short duration irradiation can induce fast depolarizations, which appear due to different mechanism. The combination of multiphoton stimulation and optical probing could enable systematic analysis of circuits.     

Theoretical analysis of thermal damage in biological tissues caused by laser irradiation

A bioheat transfer approach is proposed to study thermal damage in biological tissues caused by laser radiation. The laser light propagation in the tissue is first solved by using a robust seven-flux model in cylindrical coordinate system. The resulting spatial distribution of the absorbed laser energy is incorporated into the bioheat transfer equation for solving temperature response. Thermal damage to the tissue is assessed by the extent of denatured protein using a rate process equation. It is found that for the tissue studied, a significant protein denaturation process would take place when temperature exceeds about 53 degrees C. The effects of laser power, exposure time and beam size as well as the tissue absorption and scattering coefficients on the thermal damage process are examined and discussed. The laser conditions that cause irreversible damage to the tissue are also identified.