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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).
OM&P
Opera Med Physiol 2016 Vol. 2 (S1) 97
