Relationship between patterns of autonomous activity and external glucose concentration of culture medium
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1
Kwansei Gakuin University, Dept. of Sci. and Tech., Japan
Motivation: Cultured neuronal network on a multi electrode array (MEA) dish is useful model for electrical dynamics of neuronal network [1]. Cultured neurons autonomously form a complex network on a culture dish and spontaneous electrical activity can be observed. Spontaneous activity is generated by synaptic interaction between neurons and reflects internal states of neuronal network. The origin of the spontaneous activity is based on the electrical activity of the neurons, undertaken by active transport of ions, which requires energy from ATP. Intercellular ATP concentration is strictly controlled, however the concentration is considered to be influenced by external glucose concentration in external solution. Thus, we estimated the relationship between spontaneous activity and external glucose concentration.
Previously, we reported that, as the glucose concentration increases, the frequency of spontaneous electrical spikes increased until 15 mM concentration of the glucose in the recording solution, however it turned to decrease in the case that the glucose concentration was over 20 mM. Cultured neuronal networks were maintained in the culture medium with 17.5 mM glucose. The 17.5 mM concentration is general for culture of hippocampal neurons, and this concentration is near to 15 mM, the concentration with maximum number of spontaneous activity spikes. In addition, we found that cultured neuronal networks is most active in the solution with the glucose of the same concentration as the glucose concentration of the culture medium during maintenance. These results suggest that basic electrical background activity of cultured neuronal network is controlled by glucose concentration of culture medium during the maintenance.
So we elucidated the relationships between network activity during culture days and external glucose concentrations.
Materials and Methods: Rat hippocampal neurons were cultured on MEA (MED probe, alpha MED scientific Inc, Japan). The hippocampal region of brain was cut off from Wistar rat on embryonic day 18 and they were dissociated by 0.175 % trypsin (Life Technologies, USA). Neurons were plated on the MED probe at the density of 7800 / mm2. Culture medium consisted of 45% Ham’s F12, 45% Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies, USA), 5% horse serum (Life Technologies, USA) and 5% fatal bovine serum (Life Technologies, USA), 100 units/ml-100 μg/ml penicillin-streptomycin (Life Technologies, USA), and 5 μg/ml insulin (Sigma-Aldrich, USA). The glucose concentration of culture medium was about 17.5 mM, which is common concentration of the culture medium for neurons. In this study, culture medium contained 7.5 mM and 30 mM glucose were used for cultivating neuronal networks. Electrical activity of a living neuronal network was measured using the extracellular potential multisite recording system (MED64, Alpha MED Scientific、Japan) [2]. the cultures used for electrophysiological experiments were approximately 14 - 42 days in vitro (DIV).
Result: We calculated averaged summation of spontaneous electrical spikes from all 64 electrodes (Fig.1). In the case of culture medium with 17.5 mM glucose concentration, mean spike rates in each 1 s on DIV14, DIV21, DIV 28, DIV 35 and DIV 42 was 74.05 ± 25.39 (Mean ± SE, N = 6), 118.04 ± 16.51 (Mean ± SE, N = 6), 183.65 ± 33.96 (Mean ± SE, N = 6), 346.38 ± 30.18 (Mean ± SE, N = 6), 532.76 ± 28.27 (Mean ± SE, N = 6),respectively (Fig.1). In the case of culture medium which has 30 mM glucose concentration, mean spike rates in each 1 s on DIV14, DIV21, DIV 28, DIV 35 and DIV 42 was 27.30 ± 7.49 (Mean ± SE, N = 6), 112.94 ± 15.11 (Mean ± SE, N = 6), 252.40 ± 34.87 (Mean ± SE, N = 6), 381.00 ± 72.59 (Mean ± SE, N = 6) and 463.13 ± 67.25 (Mean ± SE, N = 6), respectively. In the case of culture medium which has 7.5 mM glucose concentration, mean spike rates in each 1 s on DIV14, DIV21, DIV 28, DIV 35 and DIV 42 was 6.94 ± 2.93 (Mean ± SE, N = 6), 54.17 ± 11.76 (Mean ± SE, N = 6), 157.94 ± 44.62 (Mean ± SE, N = 6), 217.32 ± 43.45 (Mean ± SE, N = 6) and 264.10 ± 34.12 (Mean ± SE, N = 6) respectively (Fig.1). Neurons in the cultured networks adjust the spontaneous electrical activity according to the glucose concentration of the culture medium during the maintenance.
Discussion: We confirmed that the frequency of spontaneous electrical spikes increased accompany with culture days. In the case of 30 mM glucose concentration, trend of spontaneous activity was similar to the case of 17.5 mM glucose concentration. In the case of cultivating in 7.5 mM glucose concentration, the number of spontaneous electrical spikes increased accompany with culture days similar to the case of 17.5 mM glucose concentration, however, the frequency of the spontaneous spikes was significantly low comparing with the case of 17.5 mM glucose concentration. These results indicate that neurons adjust the network spontaneous activity according to the glucose concentration of culture medium during the maintenance (and development) of the neuronal network. This may be undertaken by the density of neuronal cells adjusted during culture days.
Conclusion: We confirmed that cultured neuronal networks adjust its spontaneous network electrical external activity according to the glucose concentration of culture medium during the maintenance. The result suggests that the neuronal circuit changes their properties and adapted to the culturing environments during the maintenance.
References:
[1] Y. Jimbo, T. Tateno, and H. P. Robinson, "Simultaneous induction of pathway-specific potentiation and depression in networks of cortical neurons," Biophys J, vol. 76, pp. 670-8, Feb 1999.
[2] T. Taenaka, H. Ito,M. Murata, S.N. Kudoh, "ANN generation according to a connection map of cultured network of living neurons on a dish.," Proceedings of MHS, pp. 486-489, 2011.
Figure Legend: Fig.1 Summary of trends of mean spike rate in the case of culture medium with 17.5 mM, 7.5 mM, and 30 mM glucose concentration.
Acknowledgements
This research was supported by JSPS KAKENHI Grant Number 24300091 and MEXT-Supported Program for the Strategic Research Foundation at Private Universities S1411038.
Keywords:
spontaneous activity,
glucose metabolism,
Cultured neuronal network,
multi-electrodes array
Conference:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays, Reutlingen, Germany, 28 Jun - 1 Jul, 2016.
Presentation Type:
Poster Presentation
Topic:
MEA Meeting 2016
Citation:
Minoshima
W,
Ito
H and
Kudoh
SN
(2016). Relationship between patterns of autonomous activity and external glucose concentration of culture medium.
Front. Neurosci.
Conference Abstract:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays.
doi: 10.3389/conf.fnins.2016.93.00093
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Received:
22 Jun 2016;
Published Online:
24 Jun 2016.
*
Correspondence:
Dr. Wataru Minoshima, Kwansei Gakuin University, Dept. of Sci. and Tech., Sanda, Japan, w.minoshima@kwansei.ac.jp