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Site Updated:
10/24/2009

Bio-Engineering Research

Spatiotemporal control of neuronal activity

Objective:
Develop an all optical interface for parallel, remote, and spatiotemporal control of neuronal activity.

Background:
The human brain is an organized interconnected network of more than 100 billion nerve cells, whose activities underlie perception, thought, decision-making, and action. A primary challenge of neuroscience is to understand how groups of cells in the massive neural networks of the brain communicate and dynamically regulate their connections. As a result, there is a need for tools that permit the organized activation and monitoring of activity in groups of cells that represent discrete components of large networks.

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Figure 1. An all-optical platform for parallel and remote control of neuronal cells. Cells transfected with iGluR6 and chemically modified with the chemical photoswitch MAG to generate the light-activated ionotropic glutamate receptor (LiGluR) are loaded with calcium dye (Fluo-4) and placed on the stage of the optical microscope. Two LEDs at different wavelengths serve as sources of illumination of a DMD, which projects spatially designed patterns of the light through an objective lens onto selected cells within the field. Illumination at 380 nm opens the LiGluR channels and illumination at 505 nm closes them, thereby exciting the cells or turning the excitation off, respectively. The image sequences displayed by the DMD were synchronized with light pulses from the LEDs. Activation of the cells, detected by Fluo-4 (illuminated at 488 nm) due to calcium entering the excited cells, was imaged using a CCD.

Figure 2. Parallel optical stimulation of multiple HEK 293 cells. (a) Optical stimulation protocol. Within one optical stimulation and imaging loop, 380 and 505 nm irradiation were applied to open and close LiGluR, respectively, and two captured CCD images (before and after 380 nm irradiation), were used to determine the cell response as the difference in Fluo-4 intensity. (b) Resting Fluo-4 image of the HEK cells (scale bar) 60 um). (c) Percent Fluo-4 intensity change using flood exposure of entire field. (d) Six example frames showing accurate parallel optical stimulation. Black and white images indicate the DMD displayed patterns. Color images on right are cell responses corresponding to exposure patterns on left (Fluo-4 intensity change increases with color scale from blue to red). (e) Cell response distribution for all the stimulated cells over 33 frames using distinct illumination patterns (see Supporting Information, Movie.1). The pixel with maximum intensity change in each cell region was picked up to judge the cell response. The percentage of successful stimulation was 98.8% using the threshold of 5.1% for the Fluo-4 intensity change (P < 0.05, one-tailed t-test, n ) 99).

Figure 4. Patterned optical stimulation of cultured hippocampal neurons. (a) Optical stimulation protocol. Optical activation of neurons calculated as intensity difference between Frame 3 and Frame 1. Difference between Frame 4 and subsequent Frame 1 in next loop (LiGluR closed period) was used to determine if LiGluR activity was successfully shut off by irradiation at 505 nm. Cell calciumimages were averaged from 10 loops with the same spatialstimulation pattern and normalized to enhance signal to noise and extract optical stimulation elicited activity over spontaneous activity on the cells. (b) Resting Fluo-4 image. (c) Cell responses to flood exposure of entire field at 380 nm triggers activity in two cells (A and B). Scale bar ) 60 um for (b,c). (d-i) Magnified images of region in white box in (c). (d,e) Neuronal calcium responses whenonly cell A was optically stimulated ((d) at 380 nm; (e) at 505nm). (f,g) Neuronal calcium responses when only cell B was opticallystimulated ((f) at 380 nm; (g) at 505 nm). Neuron A had some activity when B was stimulated, but there was no response in B when A was stimulated, possibly indicating a synaptic input by B onto A. (h,i) Neuronal calcium responses when both A and B cells were optically stimulated together ((h) at 380 nm; (i) at 505 nm).

The ability to optically stimulate and detect neural activity using a device that can address multiple cells simultaneously is a significant advance over current techniques for investigating neural circuits. Now, using our optical method, light-sensitive proteins whose expression can be targeted to desired specific cell types, or caged proteins or compounds, can be activated in parallel at selective multiple sites around a cell, or at multiple cells in any desired spatiotemporal modulated manner, with the ability to rapidly switch between multiple wavelengths and illumination patterns in milliseconds. The micron-scale spatial precision of our method can potentially be used to study responses to subcellular stimulation, while the ability to precisely stimulate multiple cells within neural circuits is well suited to studies and controls of circuits within living animals that can potentially bridge the massive information flow between the man-made computer and in vivo neural circuits. Furthermore, combining the affinity labeling of MAG and micro-stereolithography may inspire a “neural lithography” leading to spatially modulating neural optical sensitivity by spatially biased labeling with patterns of light. In general, when not limited to the neural system our method of spatiotemporal uncaging chemical compounds or activating proteins can be useful for cell regulation, differentiation, and migration study and may even lead to medical and clinic applications.
Representative Paper: