Use of optogenetic technology in cell culture models, implantable device to works in slices and live animals
Brain is one of the most complicated and poorly understood parts of the human body. There is a large group of disorders related to abnormalities in brain activity called neurodegenerative diseases. At the moment etiology and pathological basis of these diseases are unknown. That’s why the fundamental assays in neurobiological field become more and more important. New technology that allows scientists to solve these biomolecular problems is called optogenetics. Optogenetics is a modern approach to modulate physiological status of excitable cells, including neurons. This modulation is achieved by combining the techniques of genetic engineering and photonics.
Cells of human embrionic kidney (line HEK-293T) are easily transfected so they are often used as an object of study. In our preliminary experiments HEK-293T cells were transfected with Channelrhodopsin and Halorhodopsin constructs. The responses were recorded using the patch-clamp technique in voltage clamp mode using blue and orange light stimulation.
After successful approbation on HEK cells, optogenetic experiments were conducted in mouse primary hippocampal neuron cultures. Neurons were transfected with ChR2-GFP plasmidand stimulated by blue (470 nm) light. Traces of neurons activity were recorded in voltage and current clamp modes.
Brain functions studying requires neuron interface that could record parameters and stimulate brain with high time-space accuracy. Most researchers who use optogenetic method in laboratory conditions on in-vivo animals now use optical fiber that is sent through the implantable cannula .
Parameters of the pulses sequence and their generation is controlled by computer graphical interface or manual switch of modes.
Programmed LED control drivers ensure the setting of DC values for one or several separate LEDs or a cluster that consists of several diodes united in one output fiber. Each channel is controlled autonomously (manually in modes of CW, external TTL or analog modulation types) or by software installed on computer.
In department of Medical Physics in our Molecular Neurodegeneration Laboratory we are working on the development and testing of implantable device for monitoring of brain neurons physiological parameters (action potential).
Together with "Nano and Microsystem Technology" research laboratory we are developing a combined optical-electrode device that allows you to carry out combined research with the use of intravital microelectrode stimulation and optogenetic activation of genetically differentiated neurons.
Optrode allows to record electrical activity during optogenetic experiments. This combination of several microelectrodes allows you to record the activity of several neurons in light affected areas. It minimizes the effects of light diffusion inside the tissue and the mismatch of positions of the light source and the detector that records neurons excitation / inhibition parameters. An implant consists of the coaxial conical optical wave guide (optrode) integrated inside the implantable electrode array (multi-electrode array-MEA) for recording the experimental data.
Alzheimer’s disease (AD) and aging are resulting in impaired ability to store memories, but the mechanisms responsible for these defects are poorly understood. It is known that electrophysiological response of mutant mouse neuron cultures in case of electrical stimulation results in significant decrease in frequency of action potentials . Our future plans include use of optogenetics in slices and live animals from AD models. For these purposes we want to use the prototype of our optrode. Our future plans also include using the device in long-term experiments on the spinal cord motor neurons stimulation using optogenetic techniques.