The fields of neurophysiology and medical bionics rely heavily on techniques that allow controllable stimulation of electrical responses in neural tissue. While electrical stimulation remains the gold standard in neural excitation, it suffers from a number of drawbacks such as the presence of stimulation artifacts when recording neural responses, and a lack of stimulation specificity due to the spread of current into surrounding tissue 1.

The last two decades have seen the development of optically mediated stimulation techniques 2. Several of these techniques require modification of the target tissue, either via the addition of a particular molecule (e.g. caged molecules) 3 or some form of genetic manipulation (e.g. optogenetics) 4, neither of which are easy to apply outside of a research setting. Of particular interest therefore is infrared neural stimulation (INS), whereby neural tissue is excited by pulsed infrared laser light. INS has the potential to overcome many of the shortcomings of electrical stimulation by enabling highly specific, non-contact stimulation of neural tissue 2. However, while INS has been successfully demonstrated in a variety of settings in vivo, the precise mechanism of excitation remains uncertain.

Some recent publications have shown progress towards uncovering the mechanism behind INS 5-7. Rapid heating due to absorption of the laser light by water appears to play a key role. However, beyond this a consensus is yet to be reached. Shapiro et al. 7 propose a highly general mechanism whereby rapid heating causes a perturbation in the distribution of charged particles adjacent to the cell membrane, leading to a change in the capacitance of the cell membrane and subsequent depolarization. In addition, Albert et al. 5 assert that laser induced heating activates a specific class of temperature sensitive ion channels (transient receptor potential vanilloid channels), allowing ions to pass through the cell membrane. At this stage it is unclear how these mechanisms combine, or indeed whether there are further factors that are yet to be identified.

Although a small number of publications (references 5,7-9) have investigated INS in vitro, the vast majority of work published in this field has been carried out in vivo (e.g. references 1,6,10-18). Infrared stimulation of auditory neurons has been an area of particular interest, owing to the potential applications in cochlear implants 10,14-18. While in vivo experiments are important to verify the effectiveness of the technique in various settings, the increased level of control afforded by in vitro studies is expected to lead to a more detailed understanding of the mechanism responsible for INS. This report describes the preparation of cultured spiral ganglion neurons for patch clamp investigations, as these can be used to study fundamental mechanisms while also linking to the large body of existing data from the auditory system.

The patch clamp technique is an excellent tool for investigations of electrophysiological phenomena, providing a means of recording electrical activity in single cells and studying the contribution of the individual underlying currents19. When this technique is applied to a stable in vitro preparation of primary neurons, such as cultured spiral ganglion neurons, it offers the opportunity to study in depth the mechanisms by which neural activity is controlled and manipulated.

The protocols specified in this work outline methods for investigating the effect of laser stimulation on the electrical properties of spiral ganglion neurons through patch clamp recordings. The approach is based on a fiber-coupled laser rather than a free-space laser, allowing safer operation as well as easier and more repeatable alignment without the need to modify the standard microscope configuration. On the basis of these protocols, it should be possible to conduct a wide range of experiments in order to more clearly determine the mechanism or mechanisms behind INS.