Optogenetic Stimulation of Human Neural Networks Using Fast Ferroelectric Spatial Light Modulator--Based Holographic Illumination  
[PERSON]  
1 Faculty of Electrical and Computer Engineering, Laboratory of Measurement and Sensor System Technique, Technische Universitat Dresden, Helmholtzstr. 18, 01069 Dresden, Germany;  
small cell samples or stimulate an unknown number of neurons simultaneously. Both patch-clamp and extracellular recording can partially overcome their limitations with the help of optogenetics. Optogenetics is a set of methods enabling control of the electrical activity of cells using light. The light sensitivity is provided by light-gated ion channels in the cell membrane, for example Channelrhodopsin-2 (ChR2) [16], which originate from green algae. These optogenetic tools are DNA-encoded and can be transduced to well-defined sets of target cells, which then express the ion channels and thus become light-sensitive. This allows for the temporally and spatially highly specific excitation of neurons and other cells either by genetically modifying specific cell types or by using focal light sources for targeted activation. Thus, optogenetic interventions not only resulted in novel medical applications like retina [17] or cochlea [18] resensitization, but also for the investigation of basic neural network functions, like signal processing and memory formation. Insights into neural network functions can mainly be gained by analyzing the kind of connections present and their meaning for certain tasks of the network as well as their temporal evolution. The most meaningful network analyses are obtained by providing a spatially and temporally well-defined input to the network and observing the network response. In the case of neural networks, the most well-defined input is the independent activation of single cells. For optimal single-cell optogenetic activation, several traits of neurons have to be taken into account. First the neuronal cell size. From general cell-size measurements, the average cell body size can be estimated to about 10 \(\upmu\)m [19]. Therefore, since the cell density in a cultured random neural network can be very high, the minimal focus of an optical system for activating these cells should at most be of this size. Second is the nature of neurons as all-or-nothing switches. This means that a single neuron fires an electrical signal if the sum of all of its inputs rises over an individual threshold. Since one neuron can have connections to many others, the ability to simultaneously stimulate several neurons can be key in investigating their connections. Since neurons fire electrical signals with lengths that are in the millisecond range, temporal control over the stimulation process has to be ensured in the millisecond range as well. As a conclusion, there are several illumination parameters the ideal setup should have for probing neuronal connections: First, achieving single cell resolution. Second, simultaneously illuminating multiple locations in an area or volume. Third, it should be possible to set time delays between illuminating single locations millisecond temporal resolution. The first requirement can be easily achieved by an illumination setup with steering mirror galvanometers or acousto-optic deflectors. These have been used for activating cardiomyocytes [20] or for two-photon excitation and imaging of neural networks [21; 22; 23]. Although these systems are capable of line scan velocities in the kilohertz range, they cannot truly generate multiple spots simultaneously. Some cells may be stimulated quasi-simultaneously by quickly switching between cells, however, the number is limited to a few by the opening time of the used ion channel [24]. Truly simultaneous stimulation is possible by actively shaping the illumination beam using spatial light modulators (SLM). Spatial light modulators are actively controllable pixelated devices that allow for the manipulation of light phase and/or intensity.