Response of primary auditory neurons to stimulation with infrared light in vitro.

OBJECTIVE: Infrared light can be used to modulate the activity of neuronal cells through thermally-evoked capacitive currents and thermosensitive ion channel modulation. The infrared power threshold for action potentials has previously been found to be far lower in the in vivo cochlea when compared with other neuronal targets, implicating spiral ganglion neurons (SGNs) as a potential target for infrared auditory prostheses. However, conflicting experimental evidence suggests that this low threshold may arise from an intermediary mechanism other than direct SGN stimulation, potentially involving residual hair cell activity. APPROACH: Patch-clamp recordings from cultured SGNs were used to explicitly quantify the capacitive and ion channel currents in an environment devoid of hair cells. Neurons were irradiated by a 1870 nm laser with pulse durations of 0.2-5.0 ms and powers up to 1.5 W. A Hodgkin-Huxley-type model was established by first characterising the voltage dependent currents, and then incorporating laser-evoked currents separated into temperature-dependent and temperature-gradient-dependent components. This model was found to accurately simulate neuronal responses and allowed the results to be extrapolated to stimulation parameter spaces not accessible during this study. MAIN RESULTS: The previously-reported low in vivo SGN stimulation threshold was not observed, and only subthreshold depolarisation was achieved, even at high light exposures. Extrapolating these results with our Hodgkin-Huxley-type model predicts an action potential threshold which does not deviate significantly from other neuronal types. SIGNIFICANCE: This suggests that the low-threshold response that is commonly reported in vivo may arise from an alternative mechanism, and calls into question the potential usefulness of the effect for auditory prostheses. The step-wise approach to modelling optically-evoked currents described here may prove useful for analysing a wider range of cell types where capacitive currents and conductance modulation are dominant.

Thermal damage threshold of neurons during infrared stimulation.

In infrared neural stimulation (INS), laser-evoked thermal transients are used to generate small depolarising currents in neurons. The laser exposure poses a moderate risk of thermal damage to the target neuron. Indeed, exogenous methods of neural stimulation often place the target neurons under stressful non-physiological conditions, which can hinder ordinary neuronal function and hasten cell death. Therefore, quantifying the exposure-dependent probability of neuronal damage is essential for identifying safe operating limits of INS and other interventions for therapeutic and prosthetic use. Using patch-clamp recordings in isolated spiral ganglion neurons, we describe a method for determining the dose-dependent damage probabilities of individual neurons in response to both acute and cumulative infrared exposure parameters based on changes in injection current. The results identify a local thermal damage threshold at approximately 60 °C, which is in keeping with previous literature and supports the claim that damage during INS is a purely thermal phenomenon. In principle this method can be applied to any potentially injurious stimuli, allowing for the calculation of a wide range of dose-dependent neural damage probabilities. Unlike histological analyses, the technique is well-suited to quantifying gradual neuronal damage, and critical threshold behaviour is not required.

Time-dependent activity of primary auditory neurons in the presence of neurotrophins and antibiotics.

In vitro cultures provide a valuable tool in studies examining the survival, morphology and function of cells in the auditory system. Primary cultures of primary auditory neurons have most notably provided critical insights into the role of neurotrophins in cell survival and morphology. Functional studies have also utilized in vitro models to study neuronal physiology and the ion channels that dictate these patterns of activity. Here we examine what influence time-in-culture has on the activity of primary auditory neurons, and how this affects our interpretation of neurotrophin and antibiotic-mediated effects in this population. Using dissociated cell culture we analyzed whole-cell patch-clamp recordings of spiral ganglion neurons grown in the presence or absence of neurotrophins and/or penicillin and streptomycin for 1-3 days in vitro. Firing threshold decreased, and both action potential number and latency increased over time regardless of treatment, whilst input resistance was lowest where neurotrophins were present. Differences in firing properties were seen with neurotrophin concentration but were not consistently maintained over the 3 days in vitro. The exclusion of antibiotics from culture media influenced most firing properties at 1 day in vitro in both untreated and neurotrophin-treated conditions. The only difference still present at 3 days was an increase in input resistance in neurotrophin-treated neurons. These results highlight the potential of neurotrophins and antibiotics to influence neural firing patterns in vitro in a time-dependent manner, and advise the careful consideration of their impact on SGN function in future studies.

Nanoparticle-enhanced infrared neural stimulation.

OBJECTIVE: Recent research has demonstrated that nerves can be stimulated by transient heating associated with the absorption of infrared light by water in the tissue. There is a great deal of interest in using this technique in neural prostheses, due to the potential for increased localization of the stimulus and minimization of contact with the tissue. However, thermal modelling suggests that the full benefits of increased localization may be reduced by cumulative heating effects when multiple stimulus sites and/or high repetition rates are used. APPROACH: Here we review recent in vitro and in vivo results suggesting that the transient heating associated with plasmon absorption in gold nanorods can also be used to stimulate nerves. MAIN RESULTS: Patch clamp experiments on cultured spiral ganglion neurons exhibited action potentials when exposed to 780 nm light at the plasmon absorption peak, while the amplitude of compound action potentials in the rat sciatic nerve were increased by laser irradiation of gold nanorods in the vicinity of the plasma membrane. Similarly, calcium imaging studies of NG108-15 neuronal cells incubated with Au nanorods revealed an increased level of intracellular calcium activity synchronized with laser exposure. SIGNIFICANCE: Given that the plasmon absorption peak of gold nanorods can be matched with the transparency window of biological tissues, these results demonstrate that nanorod absorbers hold great promise to enhance the process of infrared neural stimulation for future applications in neural prostheses and fundamental studies in neuroscience.

Gold-nanorod-assisted near-infrared stimulation of primary auditory neurons.

Infrared stimulation offers an alternative to electrical stimulation of neuronal tissue, with potential for direct, non-contact activation at high spatial resolution. Conventional methods of infrared neural stimulation (INS) rely on transient heating due to the absorption of relatively intense laser beams by water in the tissue. However, the water absorption also limits the depth of penetration of light in tissue. Therefore, the use of a near-infrared laser at 780 nm to stimulate cultured rat primary auditory neurons that are incubated with silica-coated gold nanorods (Au NRs) as an extrinsic absorber is investigated. The laser-induced electrical behavior of the neurons is observed using whole-cell patch clamp electrophysiology. The nanorod-treated auditory neurons (NR-ANs) show a significant increase in electrical activity compared with neurons that are incubated with non-absorbing silica-coated gold nanospheres and control neurons with no gold nanoparticles. The laser-induced heating by the nanorods is confirmed by measuring the transient temperature increase near the surface of the NR-ANs with an open pipette electrode. These findings demonstrate the potential to improve the efficiency and increase the penetration depth of INS by labeling nerves with Au NRs and then exposing them to infrared wavelengths in the water window of tissue.

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.

Modeling of the temporal effects of heating during infrared neural stimulation.

A model of infrared neural stimulation (INS) has been developed to allow the temporal characteristics of different stimulation parameters and geometries to be better understood. The model uses a finite element approach to solve the heat equation and allow detailed analysis of heat during INS with both microsecond and millisecond laser pulses. When compared with experimental data, the model provides insight into the mechanisms behind INS. In particular, the analysis suggests that there may be two broad regimes of INS: the process tends to be limited by the total pulse energy for pulse lengths below 100 µs, while the temperature gradient with respect to time becomes more important above 100 µs.

Effect of phase mask alignment on fiber Bragg grating spectra at harmonics of the Bragg wavelength.

Effects of fabrication conditions on the double-peak structure observed in fiber Bragg gratings at harmonics of the Bragg wavelength were investigated, showing that slight variations in the alignment of the phase mask can affect the grating spectra significantly. A single peak occurs only when the incident beam direction is perfectly normal with respect to the fiber.

Modeling of light absorption in tissue during infrared neural stimulation.

A Monte Carlo model has been developed to simulate light transport and absorption in neural tissue during infrared neural stimulation (INS). A range of fiber core sizes and numerical apertures are compared illustrating the advantages of using simulations when designing a light delivery system. A range of wavelengths, commonly used for INS, are also compared for stimulation of nerves in the cochlea, in terms of both the energy absorbed and the change in temperature due to a laser pulse. Modeling suggests that a fiber with core diameter of 200 µm and NA=0.22 is optimal for optical stimulation in the geometry used and that temperature rises in the spiral ganglion neurons are as low as 0.1°C. The results show a need for more careful experimentation to allow different proposed mechanisms of INS to be distinguished.