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Volga Neuroscience School 2016 Astroglial control of rhythm genesis in the brain
Unidirectional Axon Growth in Microchannels of Various Shapes
Y.I. Pigareva1 *, A.A. Gladkov1,2, V.N. Kolpakov1, A.S. Pimashkin1, A.Y. Bukatin3, E.I. Malishev3, I.V. Mukhina1,2 and V.B. Kazantsev1
1 Department of Neuroengineering, Center of Translational Technologies, Lobachevsky State University of Nizhny Novgorod, Nizhny Novgorod, Russia;
2 Central Research Laboratory, Cell Technology Department, Nizhny Novgorod State Medical Academy, Nizhny Novgorod, Russia;
3 Saint-Petersburg National Research Academic University of the Russian Academy of Sciences, Saint-Petersburg, Russia. * Presenting e-mail: [email protected]
Aims
Neuronal networks cultured in microfluidic chips are widely used for investigation of functional connectivity of neural culture with defined morphology [1, 2]. In this study we used hippocampal cultures plated in two chambers of microfluidic device which were connected by microchannels. We investigated various shapes of microchannels which guided axon growth in one direction between two cultures.
Methods
Microfluidic devices consist of two chambers, providing unidirectional axon growth between Source and Target neural networks (Fig. 1A). Microchannels' design was based on a sequence of various triangular segments: each segment has a "bottleneck" for reducible backward axons growth probability and convergent walls facilitated axons growing to Target chamber [2].
PDMS microfluidic chips were fabricated by two layer lithography and PDMS molding techniques. Mold design contained: first 5 |jm-thick layer, which formed microchannel structure and second 50 |jm-thick layer, which formed chambers. For mold fabrication, Silicon wafers and negative photoresists SU8 (MicroChem, USA) were used. Microchannels' structure was based on several types of segments (Fig. 1B) and their length was 600 |jm (2 - 7 segments) which is enough to provide a growth only for axons. Dissociated hippocampal neurons were plated into separate chambers.
Results
In order to find optimal design for unidirectional connectivity between neuronal sub-populations we studied axon growth dynamics in various microchannels (Fig. 1B). We proposed three types of segment shapes and each type was consist of three segment lengths of 67 |jm, 100 |jm and 200 |jm. The angle of segment's corner turned to "Target" was 45°. The diameter of "bottleneck" was 5 |jm and it could be pulled or not. Each day after plating we analyzed individual neural branches in microchannels using automated microscope system Cell-IQ.
Fig.1 (A) Design of microfluidic chip. Axons grow in microchannels from Source to Target chamber. (B) Various types of segment shapes. (C) Average distance of "backward" axons growth in relation to length of microchannel.
Volga Neuroscience School 2016 Astroglial control of rhythm genesis in the brain
For forward grow we found that in microchannels with pulled "bottleneck" axons growth in the "bottleneck" direction over some distance while in microchannels with ordinary "bottleneck" the axon can turn after entry in the segment. In the smaller segments axons pass through microchannel faster than in medium and big segments. For backward growing we found that if axons passed the "bottleneck" most probably they will grew alongside the sidewalls of the segments. For estimate an efficacy of microchannel we investigate axons growth in Target - Source direction during first five days. We measured the average number of segments that axons passed in "backward" direction. We found that "zig-zag" shaped segments with 100 |jm length segments were the most effective. In general, 67 and 100 |jm segments of all types showed similar results, while large and wide segments were least effective (Fig. 1C).
Conclusions
In this study we investigate microfluidic chips consists of two chambers with neuronal populations coupled by various microchannels wherein axons can grow. We found specific features of axon dynamics during growth in the segments of microchannels and found optimal design among proposed. In further study we plan to collect statistical data for axon growth and measure unidirectional efficacy of each microchannel.
Acknowledgement
The research is supported by the Russian Science Foundation (grant 14-19-01381). References
1. Habibey R., Golabchi A., Latifi S., Difato F., Blau A. (2015). Microchannel device for selective laser dissection, long-term microelectrode array electrophysiology and imaging of confined axonal projections. Lab Chip, 15(Lmd), 4578-4590.
2. Malishev E., Pimashkin A., Gladkov A., Pigareva Y., Bukatin A., Kazantsev V., Mukhina I., Dubina M. (2015). Microfluidic device for unidirectional axon growth. Journal of Physics: Conference Series, 643, 012025
The Implementation of the Cost-Effective and Adaptive Two-Photon Microscope for Neuroscience
A.V. Popov*, M.S. Doronin, Y.V. Dembitskaya, A.V. Semyanov
Institute of Neuroscience, Lobachevsky State University of Nizhny Novgorod,Nizhny Novgorod, Russia. * Presenting e-mail: [email protected]
Two-photon microscopy plays an important role in studies of the brain functioning. Particularly, it enables to visualize with a high and spatial resolution such crucial processes of neuronal functioning as changes of membrane potential (voltage-sensitive imaging) or alterations of calcium concentration (calcium imaging). Additionally, two-photon microscopy produces a relatively low photodamage to the tissue and allows to conduct relatively long measurements without significantly affecting the cell functioning not only in vitro, but also in vivo. For the last two decades, two-photon microscopy served a key role in the progress of neuroscience. However, commercial versions of microscopes usually are expensive and difficult to adapt for highly specific tasks. The commercially build software that controls the parts on a two-photon system has great limitations for rearrangements and adaptation for a particular experimental tasks as well as the hardware of such systems.
Therefore, our goal was to create a custom two-photon microscope, equipped with two adjustable two-photon femtosecond lasers (680 - 1080 nm) controlled by custom-made software. That is enable us to visualize neurons with high spatial-temporal characteristics and to stimulate locally with the high precision individual synapses (dendritic spines) by glutamate uncaging, e.g. MNI-caged-L-glutamate. Additionally, that provide us with a direct access to the modification and easy access to the program code, that can be quickly adjusted to our specific tasks, such as monitoring of local calcium events in neurons and astrocytes, which requires high sensitivity and efficiency of the system. In order to achieve that, the localization of the photomultiplier was optimized by placing it maximally close to the objective, that greatly reduces the number of lost photons and represents a distinct feature of this microscope. Another a key feature of this microscope is the custom-made software, that is highly adjustable and has been optimized for specific tasks in vitro and in vivo.
As a result , the custom-made two-photon microscope represents a highly efficient, purpose-built and cost-effective system, that is remarkably useful for conducting experimental procedures in vitro and in vivo in order to investigate the brain functioning.
Acknowledgements
This work was supported by the Russian Science Foundation (grant 16-14-00201).
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