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Volga Neuroscience School 2016 Astroglial control of rhythm genesis in the brain
Use of Optogenetic Technology in Cell Culture Models, Implantable Device to Works in Slices and Live Animals
A.I. Erofeev1,2, O.A. Zakharova1,2, M.V. Matveev2, S.G. Terekhin1,2, I.E. Eezprozvanny1,3 ,O.L. Vlasova1'2 *
1 Molecular Neurodegeneration Lab, Peter the Great Polytechnic University, Saint-Petersburg,195251, Russia.
2 Department of Medical Physics, Peter the Great St. Petersburg Polytechnic University, Saint-Petersburg, 195251, Russia.
3 Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX, USA. * Presenting e-mail: olvlasova@yandex.ru
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Abstract. Optogenetic is a powerful method that allows to modulate cellular physiological properties. In our article we demonstrate changes of electrical properties of cellular membranes on HEK-293T and hippocampal neurons transfected with channelrhodopsins and halorhodopsins induced by blue and orange light stimulation. In recent years, a method of developmental research has proved its effectiveness in the nerve cell stimulation tasks. In our article we demonstrate an implanted device for the stimulation of neurons in slices and live animals.
Introduction
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 op-togenetics. 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 [1, 2, 3].
Optogenetic approach in HEK cells
Cells of human embrionic kidney (line HEK-293T) are easily transfected so they are often used as an object of study[4]. 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 (depolarisation, Fig. 1,A) and orange (hyperpolarisation, Fig. 1, B) light stimulation.
Optogenetic approach in hippocampal neurons optical-electrode interface
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.
- - - - - A
Channel rhodopsin-2
B
Halorhodopsin wW^^^Wum/^ WpA |_ls
Fig. 1. (A, B, C). (A) Depolarization of the cell membrane during the optogenetic stimulation of transfected by ChR2-GFP plasmids cells linesHEK-293(Aex = 470 nm); (B) Hyperpolarization of the cell membrane during the optogeneticstimulation of transfected by HR-GFP plasmids cells linesHEK-293 (Aex = 590 nm); (C) confocal microscopy of transfected cell lines HEK-293T.
SOpa lis
Volga Neuroscience School 2016 Astroglial control of rhythm genesis in the brain
Optical-electrode interface
Brain functions studying requires neuron interface that could record parameters and stimulate brain with high timespace 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 [5].
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).
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Fig.2. (A, B, C, D). (A) Hippocampal neurons expressing ChR2-GFP (scale bar 20 ym); (B) Hippocampal neuron culture (DIV15); (C) Inward current in voltage-clamped neuron evoked by 470 nmlight (indicated by blue bar); (D) Voltage traces showing response to light stimulation.
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 (Fig. 3).
Optrode allows to record electrical activity during optogenetic experiments. This combination of several mi-croelectrodes 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. [6] 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.
Fig.3. Schematic representation of an implantable optical-electronic array: 1 - the body of the array; 2 -integrated optrode; 3 -multichannel electrical data recording; 4 - electrode needle; 5 - optogenetic control signal.
Volga Neuroscience School 2016 Astroglial control of rhythm genesis in the brain Future plans and conclusions
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 [7]. 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.
Acknowledgments
This work was supported by the state grant 17.1360.2014/K, by the Russian Scientific Fund grant 14-25-00024: The financial support was divided in the following way: experiments depicted on Figure 1 was supported by the state grant 17.1360.2014/K, experiments depicted on Figure 2 was supported by the Russian Scientific Fund grant.
References
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2. Boyden ES, Zhang F, Bamberg E, Nagel G, Deisseroth K 2005 Nat. Neurosci.8(9) 1263
3. Windhorst U, Johansson H1999Springer 190
4. He B, Soderlund D M 2010 Neuroscience Letters469(2) 268
5. Sparta D.R., Stamatakis A.M., Phillips J.L., et al., 2012, 7(1) 12.
6. Anikeeva P., Andalman A.S., Witten I., et al., 2011, Nature Neuroscience 15(1) 163.
7. Zhang H, Jie Liu J, Sun S, Pchitskaya E, Popugaeva E and Bezprozvanny I2015Journal of Alzheimer's Disease (45) 561
Complex Behaviour in Cyclic Competition Bimatrix Games
Cezary Olszowiec*
Departament de Fisica, Universitat Politecnica de Catalunya, Terrassa, Barcelona, Spain. * Presenting e-mail: c.olszowiec14@imperial.ac.uk
We consider an example of cyclic competition bimatrix game which is a Rock-Scissors-Paper game with perfect memory of the playing agents.
At first we investigate the dynamics in the neighbourhood of the Nash equilibrium as well as the dynamics on the boundary of codimension 1 - that is when one of the strategies is not played by any agent.
For the analysis of asymptotic behaviour close to the boundary, we provide the description of naturally appearing heteroclinic network, with discretized dynamics in its neighbourhood. Quotient network is investigated as well and compared to the invalid models already considered in the literature. It turns out that certain types of behaviour are never possible or appear in the system only for some parameter values. Moreover, parameter space is divided into four regions where we observe either chaos, or preference to follow itinerary consisting of strategies for which one or the other agent do not lose, or they alternate in winning.
These regions are separated by two analytical curves and lines where game is either symmetric (system is not C1 linearisable) or is zero-sum (so the system is Hamiltonian).
On each of these curves we observe different bifurcation scenarions: e.g. transition from order to chaos, or from one kind of stability to other one, or just loss of one dimension of the local stable manifold of the subcycle.
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