Selective Optical Stimulation
of Neurons grown on Si wafers

We have developed a new technique for selective extracellular stimulation of neurons in culture using targeted illumination pulses. We are currently utilizing this stimulation technique for studying collective properties of small neural networks in-vitro. Focused laser beam is used to selectively stimulate any location of a cultured 2D neural network in the microscope field of view. This offers an advantage over Multi-Electrode Array approach (MEA), where only a fixed set of stimulation locations is available.

  

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Results:

Detailed description of the technique was presented here. We used patch clamping to charachterise the capabilities and limitations of this type of extracellular stimulation. Spikes and subthreshold events in a single neuron were intracellularly recorded while pulsing the laser in specific locations of neural network. General purpose hardware and software pieces used for data aquisition in our lab are beautifully covered by Naveen here and snapshots of specialized "Optostim" GUI are shown in this page.

Green circles in the Figure correspond to automatically scanned grid and are filled white for locations where the patched neuron responded with an action potential spike (AP) in response to a laser pulse. These highly reproducible APs were antidromically stimulated in two different low-threshold areas of axonal tree: (x=500,y=350) and (200,170). Orange circles are the locations tested in point-and-click mode. A single captured intracellular voltage trace is displayed in the top-right corner. Note the stimulation artifact at 1ms point in time and an action potential spike of the patched neuron 10ms later. This response was produced when pulsing the laser at location (200,160). Check the individual voltage traces caputured in point-and-click mode, where colors refer to markers on the 2D-scan delays map. These recordings were made with synaptic transmission blocked.

A more complex mixture of sub- and suprathreshold events is observed with synaptic transmissions enabled. Note a huge 26ms delay of the synaptically transmitted AP after the stimulation at location (400,450).

Fluorescent labeling of cell processes confirm the antidromic stimulation hypothesis. All three sensitive areas in the figure are seen centered along the axon that extends clockwise around the cell. Differences in delays of APs recorded at the soma are consistent with the travel distance.

Technical details:

Neurons are grown on Silicon wafers. Voltage bias is applied between the wafer and the extracellular solution so that the interface is depleted of majority carriers: no electric current flows in dark. By pulsing the laser near a targeted neuron we generate electric carriers allowing local phtocurrent pulse across the interface. This is equivalent to instantaneously creating an extracellular electrode in the desired location. Applied bias value seems to be the major parameter for stimulation strength control, whereas the amount of light should be minimized for better selectivity.

Two Acousto-Optic Deflectors (AOD) are used to aim the laser beam faster than a typical time of the neuronal response. Different locations can be addressed almost simultaneously with a single beam. Complex spatio-temporal stimulation patterns in the field of view are possible. The dashed red line in the figure shows the laser beam path outside the optical fiber.

 

 

Last Updated on 01-Jun-2005 Contact: Artem Starovoytov