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Antibiotic resistance is a growing concern that is driving the exploration of alternative ways of killing bacteria. Here we show that gold nanoparticles synthesized by the mycelium of Mucor plumbeus are an effective medium for antimicrobial photodynamic therapy (PDT). These particles are spherical in shape, uniformly distributed without any significant agglomeration, and show a single plasmon band at 522&ndash, 523 nm. The nanoparticle sizes range from 13 to 25 nm, and possess an average size of 17 &plusmn, 4 nm. In PDT, light (from a source consisting of nine LEDs with a peak wavelength of 640 nm and FWMH 20 nm arranged in a 3 &times, 3 array), a photosensitiser (methylene blue), and oxygen are used to kill undesired cells. We show that the biogenic nanoparticles enhance the effectiveness of the photosensitiser, methylene blue, and so can be used to kill both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria. The enhanced effectiveness means that we could kill these bacteria with a simple, small LED-based light source. We show that the biogenic gold nanoparticles prevent fast photobleaching, thereby enhancing the photoactivity of the methylene blue (MB) molecules and their bactericidal effect.

Optical stimulation is a paradigm-shifting approach to modulating neural activity that has the potential to overcome the issue of current spread that occurs with electrical stimulation by providing focused stimuli. But optical stimulation either requires high power infrared light or genetic modification of neurons to make them responsive to lower power visible light. This work examines optical activation of auditory neurons following optogenetic modification via AAV injection in two species (mouse and guinea pig). An Anc80 viral vector was used to express the channelrhodopsin variant ChR2-H134R fused to a fluorescent reporter gene under the control of the human synapsin-1 promoter. The AAV was administered directly to the cochlea (n = 33) or posterior semi-circular canal of C57BL/6 mice (n = 4) or to guinea pig cochleae (n = 6). Light (488 nm), electrical stimuli or the combination of these (hybrid stimulation) was delivered to the cochlea via a laser-coupled optical fibre and co-located platinum wire. Activation thresholds, spread of activation and stimulus interactions were obtained from multi-unit recordings from the central nucleus of the inferior colliculus of injected mice, as well as ChR2-H134R transgenic mice (n = 4). Expression of ChR2-H134R was examined by histology. In the mouse, transduction of auditory neurons by the Anc80 viral vector was most successful when injected at a neonatal age with up to 89% of neurons transduced. Auditory neuron transductions were not successful in guinea pigs. Inferior colliculus responses to optical stimuli were detected in a cochleotopic manner in all mice with ChR2-H134R expression. There was a significant correlation between lower activation thresholds in mice and higher proportions of transduced neurons. There was no difference in spread of activation between optical stimulation and electrical stimulation provided by the light/electrical delivery system used here (optical fibre with bonded 25 µm platinum/iridium wire). Hybrid stimulation, comprised of sub-threshold optical stimulation to ‘prime’ or raise the excitability of the neurons, lowered the threshold for electrical activation in most cases, but the impact on excitation width was more variable compared to transgenic mice. This study demonstrates the impact of opsin expression levels and expression pattern on optical and hybrid stimulation when considering optical or hybrid stimulation techniques for neuromodulation.

Electrical stimulation has been used for decades in devices such as pacemakers, cochlear implants and more recently for deep brain and retinal stimulation and electroceutical treatment of disease. However, current spread from the electrodes limits the precision of neural activation, leading to a low quality therapeutic outcome or undesired side-effects. Alternative methods of neural stimulation such as optical stimulation offer the potential to deliver higher spatial resolution of neural activation. Direct optical stimulation is possible with infrared light, while visible light can be used to activate neurons if the neural tissue is genetically modified with a light sensitive ion channel. Experimentally, both methods have resulted in highly precise stimulation with little spread of activation at least in the cochlea, each with advantages and disadvantages. Infrared neural stimulation does not require modification of the neural tissue, but has very high power requirements. Optogenetics can achieve precision of activation with lower power, but only in conjunction with targeted insertion of a light sensitive ion channel into the nervous system via gene therapy. This review will examine the advantages and limitations of optical stimulation of neural tissue, using the cochlea as an exemplary model and recent developments for retinal and deep brain stimulation.

Electrical stimulation has long been the most effective strategy for evoking neural activity from bionic devices and has been used with great success in the cochlear implant to allow deaf people to hear speech and sound. Despite its success, the spread of electrical current stimulates a broad region of neural tissue meaning that contemporary devices have limited precision. Optical stimulation as an alternative has attracted much recent interest for its capacity to provide highly focused stimuli, and therefore, potentially improved sensory perception. Given its specificity of activation, optical stimulation may also provide a useful tool in the study of fundamental neuroanatomy and neurophysiological processes. Areas covered: This review examines the advances in optical stimulation - infrared, nanoparticle-enhanced, and optogenetic-based - and its application in the inner ear for the restoration of auditory function following hearing loss. Expert opinion: Initial outcomes suggest that optogenetic-based approaches hold the greatest potential and viability amongst optical techniques for application in the cochlea. The future success of this approach will be governed by advances in the targeted delivery of opsins to auditory neurons, improvements in channel kinetics, development of optical arrays, and innovation of opsins that activate within the optimal near-infrared therapeutic window.

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.

Objective. Established guidelines for safe levels of electrical stimulation for neural prostheses are based on a limited range of the stimulus parameters used clinically. Recent studies have reported particulate platinum (Pt) associated with long-term clinical use of these devices, highlighting the need for more carefully defined safety limits. We previously reported no adverse effects of Pt corrosion products in the cochleae of guinea pigs following 4 weeks of electrical stimulation using charge densities far greater than the published safe limits for cochlear implants. The present study examines the histopathological effects of Pt within the cochlea following continuous stimulation at a charge density well above the defined safe limits for periods up to 6 months. Approach. Six cats were bilaterally implanted with Pt electrode arrays and unilaterally stimulated using charge balanced current pulses at a charge density of 267 μC cm−2 phase−1 using a tripolar electrode configuration. Electrochemical measurements were made throughout the implant duration and evoked potentials recorded at the outset and on completion of the stimulation program. Cochleae were examined histologically for particulate Pt, tissue response, and auditory nerve survival; electrodes were examined for surface corrosion; and cochlea, brain, kidney, and liver tissue analysed for trace levels of Pt. Main results. Chronic stimulation resulted in both a significant increase in tissue response and particulate Pt within the tissue capsule surrounding the electrode array compared with implanted, unstimulated control cochleae. Importantly, there was no stimulus-induced loss of auditory neurons (ANs) or increase in evoked potential thresholds. Stimulated electrodes were significantly more corroded compared with unstimulated electrodes. Trace analysis revealed Pt in both stimulated and control cochleae although significantly greater levels were detected within stimulated cochleae. There was no evidence of Pt in brain or liver; however, trace levels of Pt were recorded in the kidneys of two animals. Finally, increased charge storage capacity and charge injection limit reflected the more extensive electrode corrosion associated with stimulated electrodes. Significance. Long-term electrical stimulation of Pt electrodes at a charge density well above existing safety limits and nearly an order of magnitude higher than levels used clinically, does not adversely affect the AN population or reduce neural function, despite a stimulus-induced tissue response and the accumulation of Pt corrosion product. The mechanism resulting in Pt within the unstimulated cochlea is unclear, while the level of Pt observed systemically following stimulation at these very high charge densities does not appear to be of clinical significance.

Infrared light can be used to modulate the activity of neuronal cells with broad generality and without any need for exogenous materials. The action potential response has been shown to be associated with heating due to the absorption of light by water in and around the illuminated tissues, which gives rise to at least two distinct processes: namely, the temperature pulses cause depolarizing capacitive currents due to an intramembrane thermo-mechanical effect, and in addition, temperature-sensitive TRPV ion channels (and likely, voltage-gated channels) drive additional membrane depolarization. However, substantial differences between the activation threshold of primary auditory neurons ( 300 mJ/cm^2) in vivo have generated some controversy in the field. A temperature-dependent Hodgkin-Huxley type model, which combines capacitive currents and the experimentally-derived characteristics of voltage-gated potassium and sodium ion channels in primary auditory neurons, was used to accurately explain the in vitro response to 1870 nm infrared illumination. TRPV channels do not make a significant contribution in this case, suggesting that the detailed mechanism of the neuronal response to infrared light is dependent on the specific cell type. Furthermore, based on this detailed understanding of the cell behaviour, it is shown that action potentials cannot be generated at safe laser power levels. This suggests that the previously reported response of the auditory system to infrared stimulation in vivo might arise from a different mechanism, and calls into question the potential usefulness of the effect for auditory prostheses.

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.

At present there is some debate as to the processes by which infrared neural stimulation (INS) activates neurons in the cochlea, as the lasers used for INS can potentially generate a range of secondary stimuli e.g. an acoustic stimulus is produced when the light is absorbed by water. To clarify whether INS in the cochlea requires functioning hair cells and to explore the potential relevance to cochlear implants, experiments using INS were performed in the cochleae of both normal hearing and profoundly deaf guinea pigs. A response to laser stimulation was readily evoked in normal hearing cochlea. However, no response was evoked in any profoundly deaf cochleae, for either acute or chronic deafening, contrary to previous work where a response was observed after acute deafening with ototoxic drugs. A neural response to electrical stimulation was readily evoked in all cochleae after deafening. The absence of a response from optical stimuli in profoundly deaf cochleae suggests that the response from INS in the cochlea is hair cell mediated.

Our capacity to interface with the nervous system remains overwhelmingly reliant on electrical stimulation devices, such as electrode arrays and cuff electrodes that can stimulate both central and peripheral nervous systems. However, electrical stimulation has to deal with multiple challenges, including selectivity, spatial resolution, mechanical stability, implant-induced injury and the subsequent inflammatory response. Optical stimulation techniques may avoid some of these challenges by providing more selective stimulation, higher spatial resolution and reduced invasiveness of the device, while also avoiding the electrical artefacts that complicate recordings of electrically stimulated neuronal activity. This review explores the current status of optical stimulation techniques, including optogenetic methods, photoactive molecule approaches and infrared neural stimulation, together with emerging techniques such as hybrid optical-electrical stimulation, nanoparticle enhanced stimulation and optoelectric methods. Infrared neural stimulation is particularly emphasised, due to the potential for direct activation of neural tissue by infrared light, as opposed to techniques that rely on the introduction of exogenous light responsive materials. However, infrared neural stimulation remains imperfectly understood, and techniques for accurately delivering light are still under development. While the various techniques reviewed here confirm the overall feasibility of optical stimulation, a number of challenges remain to be overcome before they can deliver their full potential.

Chicken parathyroid hormone (cPTH) has been reported to stimulate adrenal steroidogenesis and to have unusual potency on traditional PTH target tissues. To evaluate these properties, chicken PTH-(1-88) has been expressed in Escherichia coli using a plasmid encoding a fusion protein which links together growth hormone, a factor Xa recognition site, and chicken PTH-(1-88). The growth hormone-cPTH fusion protein required the presence of 0.02% sodium dodecyl sulfate to remain in solution and be cleaved by factor Xa. The high performance liquid chromatography-purified recombinant cPTH-(1-88) and chemically synthesized cPTH-(1-34) had similar potency in rat osteosarcoma (ROS 17/2.8) cells, opossum kidney (OK) cells, and dispersed primary chicken kidney cells. The biologic potencies of cPTH-(1-34) and cPTH-(1-88) in radioreceptor binding and cAMP generation in both bone- and kidney-derived cell lines were less than those of human (h)PTH-(1-34). In dispersed chicken kidney cells, cAMP production by cPTH-(1-34) and cPTH-(1-88) was similar to that stimulated by human PTH-(1-34). No stimulation of steroidogenesis could be detected when recombinant chicken PTH-(1-88) was added to dispersed chicken adrenal cells. The biologic activity of recombinant chicken PTH-(1-88) purified from E. coli was comparable with that of chicken PTH-(1-88) expressed by mammalian COS cells. Thus, the full-length chicken PTH did not exhibit enhanced potency, when compared with human PTH in ROS 17/2.8, OK cell lines, and dispersed chicken kidney cells and did not demonstrate the novel steroidogenic action previously reported in adrenal cells. The successful production of chicken PTH-(1-88) will enhance our understanding of the structure-activity relationships for PTH, particularly the sequence-dependent metabolism of the hormone.