Abstract
We present a method for achieving temporally and spatially precise photoactivation of neurons without the need for genetic expression of photosensitive proteins. Our method depends upon conduction of thermal energy via absorption by chemically inert carbon particles and does not require the presence of voltage-gated channels to create transmembrane currents. We demonstrate photothermal initiation of action potentials in Hirudo verbana neurons within an intact ganglion and of transmembrane currents in Xenopus oocytes. Thermal energy is delivered by focused 50 ms, 650 nm laser pulses with total pulse energies between 250 and 3500 μJ. We document an optical delivery system for targeting specific neurons that can be expanded for multiple target sites. Our method achieves photoactivation reliably (70 - 90% of attempts) and can issue multiple pulses (6-9) with minimal changes to cellular properties as measured by intracellular recording. Direct photoactivation presents a significant step towards all-optical analysis of neural circuits in animals such as Hirudo verbana where genetic expression of photosensitive compounds is not feasible.
Highlights
The use of genetically expressed photosensitive channels in neurons has dramatically expanded the ability to generate spatially and temporally precise stimuli for probing neural circuit functions.[1]
We found that irradiation of a carbon particle on the surface of a neuron with 50 ms, 250 - 700 μJ laser pulses induced action potentials in all types of neurons sampled, which included Retzius (Rz), AP, P, and T cells (Figure 2(a)[i])
We conclude that the most likely cause for photoactivation of neurons is an increase in the local temperature of the membrane
Summary
The use of genetically expressed photosensitive channels in neurons has dramatically expanded the ability to generate spatially and temporally precise stimuli for probing neural circuit functions.[1] Traditional methods require the use of stimulating electrodes that are manually fixed in a static position during an experiment. By replacing electrodes with optical methods, stimulation patterns can be dynamically changed[2] or performed in awake behaving animals.[3] In the expanding body of research using photoactivation of neurons, most methods rely on genetic expression of specific channels.[1,4] in any of the animal models such as Hirudo where genetic engineering is difficult,[5] an alternative method would be very useful for investigating the remarkable and complex behaviors found at the circuit level.[6,7] Voltage-sensitive dyes allow recording from many leech neurons simultaneously, revealing behavioral activity patterns.[8] As advances in optical recording of leech neuronal potentials increase the temporal and spatial resolution of optical electrophysiology,[9] the need for a method of photoactivating neurons to affect neuronal activity patterns has become more crucial. Such a method would allow investigators to initiate “fictive” behaviors (in which motor patterns are exhibited in the isolated nervous system)[10] via dynamic and physiologically relevant stimuli
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