Optimization of asymmetric microchannels for unidirectional synaptic coupling of neuronal cultures with high efficiency

Event Abstract Back to Event Optimization of asymmetric microchannels for unidirectional synaptic coupling of neuronal cultures with high efficiency Arseniy Gladkov1, 2*, Yana Pigareva1, Vladimir Kolpakov1, Anton Bukatin3, Victor Kazantsev1, Irina Mukhina1, 2 and Alexey Pimashkin1 1 N. I. Lobachevsky State University of Nizhny Novgorod, Russia 2 Nizhny Novgorod State Medical Academy, Russia 3 Saint Petersburg Academic University (RAS), Russia Motivation Neuronal tissue engineering using microfluidic chips combined with microelectrode arrays is a perspective approach to study fundamental problems in neurobiology. In particular, microfluidic microchannels can isolate cell soma of one cultured network from its axons which then grow and form synaptic connections with other neural culture. Such approach is used to study interaction of the cells from various regions [1,2,3]. Neural networks in vivo have unidirectional architecture and layered structure which is necessary for information transfer and processing. In our previous study we proposed a simple design for asymmetric microchannels which provided unidirectional axon growth between two neural cultures. In this study we present a microfluidic channels with asymmetry over the longitudinal axis which provide significantly higher efficiency in unidirectional axon growth. Methods Hippocampal neuronal cells from mice embryos at 18-th prenatal day were cultured within microfluidic chips combined with 60-electrode arrays (Multichannel systems, Germany) (fig. 1a). The initial cellular density was approximately 8,000-9,000 cells/mm2. Microfluidic chips were made from a biocompatible polydimethylsiloxane (PDMS). The chips consisted of two chambers for the cells and microchannels between the chambers. Axons grew through the microchannels and formed a connection between two populations of neurons. Microchannels consisted of asymmetric sections (fig. 1c). Unidirectional growth of axons was provided due to the asymmetric shape of microchannels and the bottlenecks. The cultured subnetworks were defined as the Source and the Target networks, according to the predefined direction of axon growth. We estimated the fraction of spontaneous bursts generated in one chamber and evoked burst in another chamber. To quantify the microchannel efficiency to propagate the spiking patterns in the desired direction from the Source culture to the Target culture, we defined a directional propagation index (DPI). The DPI was calculated as the ratio of the percentage of Source bursts followed by Target bursts to the percentage of Target bursts followed by Source bursts. We compared the efficiency of two types of chips with different microchannel shapes. The first type of chips had 8 microchannels. Each microchannel consisted of three identical triangular segments. This type of channel we called a narrow triangular [4]. The second type of chip had microchannels consisting of narrow straight part and a wide section with traps. This type of channel we called a wide asymmetric. Two populations of neuronal cells were connected by axons in microchannels of both types, narrow triangular (fig. 1b) and wide asymmetric (fig. 1c). Schematic representation of axonal growth in microchannels is shown in Figure 1 e, f. In contrast to previous studies, we proposed the asymmetry along the microchannel. The unidirectional connection between two neuronal cultures was achieved using microchannels with a wide section near the Target culture. 46% of spontaneous bursts generated in Chamber A evoked a burst in Chamber B. In the same time, only 2% of bursts generated in Chamber B evoked a burst in Chamber A. In narrow triangular microchannels more than 10% of the bursts generated in Chamber B evoked a burst in Chamber A [4]. DPI for wide asymmetric microchannels was 20,7 which was much higher than for narrow triangular microchannels (fig. 2). Consequently, wide asymmetric type of microchannels was more effective for the formation of a unidirectional connection between isolated neuronal cultures. Discussion The new wide asymmetric design of the microchannels was developed according to the following working hypothesis. The growth of axons towards the opposite chamber is faster in narrow segments than in the wide segment. As a result, the axons from the Source culture pass through the bottlenecks earlier then axons from the Target culture. Axons block the bottlenecks of microchannels when pass through them. Thus, the axons from the Target culture can not grow to the Source culture, even if they grow along axons from the Source culture. The developed design is highly applicable for engineering of complex neuronal circuits. Presumably the developed shape of the microchannels can be changed without decreasing the efficiency of unidirectional connectivity, for example, by bending a microchannel. In this case, the key characteristics of the microchannel must be preserved. In addition, in a wide section there is enough space for branching of the processes and localization of synaptic contacts. Conclusion We developed a new optimal design of microfluidic chip for growing unidirectional connectivity in neuronal cultures. The microchannel shape had the asymmetry of sections along the microchannel longitudinal axis. This effectively prevented the growth of axons in the opposite direction resulting in the high efficiency of unidirectional connectivity in neuronal cultures. Figure 1. Microfluidic chips for unidirectional network connectivity formation. a Microfluidic chip, combined with microelectrode array. b. Neuronal culture in a chip with microchannels composed of three narrow triangular segments. c. Neuronal culture in a chip with microchannels composed of narrow straight part and a wide section with traps (wide asymmetric). d. Scheme of neurite growth in microchannels corresponding to (b). e. Scheme of neurite growth in microchannels corresponding to (c). Figure 2. Propagation of bursting activity between two sub-networks in a chip with microchannels with a wide section (wide asymmetric). a. Raster plot of a burst propagated from Chamber A to Chamber B. b. Raster plot of a burst propagated from Chamber B to Chamber A. Firing rate profiles of bursts propagated from Chamber A to Chamber B (c) and from Chamber B to Chamber A (d). Figure 1 Figure 2 Acknowledgements This research was supported by the grant of the president of Russian Federation MК-6795.2018.4 References 1. Le Feber J, Postma W, de Weerd E, Weusthof M, Rutten WLC. Barbed channels enhance unidirectional connectivity between neuronal networks cultured on multi electrode arrays. Front Neurosci. 2015;9(10):412. 2. Pan L, Alagapan S, Franca E, Leondopulos SS, DeMarse TB, Brewer GJ, et al. An in vitro method to manipulate the direction and functional strength between neural populations. Front Neural Circuits [Internet]. 2015;9:1–14. Available from: http://journal.frontiersin.org/article/10.3389/fncir.2015.00032 3. Poli D, Thiagarajan S, DeMarse TB, Wheeler BC, Brewer GJ. Sparse and Specific Coding during Information Transmission between Co-cultured Dentate Gyrus and CA3 Hippocampal Networks. Front Neural Circuits. 2017;11(March):1–14. 4. Gladkov A, Pigareva Y, Kutyina D, Kolpakov V, Bukatin A, Mukhina I, et al. Design of Cultured Neuron Networks in vitro with Predefined Connectivity Using Asymmetric Microfluidic Channels. Sci Rep. Springer US; 2017;(10):1–14. Available from: http://dx.doi.org/10.1038/s41598-017-15506-2 Keywords: Neural Engineering, Axon navigation, Synaptic connectivity, microelectrode arrays, Microfluidic chip Conference: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays, Reutlingen, Germany, 4 Jul - 6 Jul, 2018. Presentation Type: Poster Presentation Topic: Microphysiological systems Citation: Gladkov A, Pigareva Y, Kolpakov V, Bukatin A, Kazantsev V, Mukhina I and Pimashkin A (2019). Optimization of asymmetric microchannels for unidirectional synaptic coupling of neuronal cultures with high efficiency. Conference Abstract: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays. doi: 10.3389/conf.fncel.2018.38.00005 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 18 Mar 2018; Published Online: 17 Jan 2019. * Correspondence: Mr. Arseniy Gladkov, N. I. Lobachevsky State University of Nizhny Novgorod, Nizhny Novgorod, Russia, arseniy.gladkov@yahoo.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Arseniy Gladkov Yana Pigareva Vladimir Kolpakov Anton Bukatin Victor Kazantsev Irina Mukhina Alexey Pimashkin Google Arseniy Gladkov Yana Pigareva Vladimir Kolpakov Anton Bukatin Victor Kazantsev Irina Mukhina Alexey Pimashkin Google Scholar Arseniy Gladkov Yana Pigareva Vladimir Kolpakov Anton Bukatin Victor Kazantsev Irina Mukhina Alexey Pimashkin PubMed Arseniy Gladkov Yana Pigareva Vladimir Kolpakov Anton Bukatin Victor Kazantsev Irina Mukhina Alexey Pimashkin Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.