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Neuronal network synchronization is mediated through astrocytic glutamate transporters

Ravi Kumar1, 2*, Shun-Fen Tzeng3 and Chi-Keung Chan2, 4
  • 1 Taiwan International Graduate Program in Interdisciplinary Neuroscience, National Cheng Kung University and Academia Sinica, Taiwan
  • 2 Institute of Physics, Academia Sinica, Taiwan
  • 3 Department of Life Sciences, National Cheng Kung University, Taiwan
  • 4 Graduate Institute of Biophysics, National Central University, Department of Physics, Taiwan

Motivation Cognitive processes are related with orchestrated synchronization and propagation of firing activities among neuronal ensembles. The synchronized firings occur in the form of synchronized burst-firings, also referred as Network Spikes (NS), which is initiated by sequential activation of cell assemblies (1). NS are often observed in vivo as well as in vitro conditions. In vivo, NS have been seen in physiological; visual system development (2), memory formation, integration of sensory inputs, as well as in pathological brain conditions; epilepsy (3), tremors. The origin of such events is still limited to the plausible notion of balance between excitatory and inhibitory inputs among neurons in the circuit level. However, recent research on neuron-astrocyte tripartite interaction at the synaptic level (4) indicate that a neuro-glial networking could be the possible mechanism for the network level synchronization. Here, we show that pharmacological blocking of astrocytic GLT-1 glutamate transporters in cortical cultures disrupts network synchrony reversibly. Materials and methods Primary cortical cells were extracted from mice postnatal pups (P0) and plated (~2000 cells/mm2) on planar 4-well multi electrodes arrays (MEA60-4Well-PtB, Qwane Biosciences SA, Switzerland). The cultured cells were maintained in NeuroBasal-A medium (Invitrogen) inside humidified incubator with 5% CO2 at 37∘C. Half of their media were periodically replaced with fresh media in every 3 days. Electrical activities from the developed cortical networks were amplified and recorded using in vitro MEA-systems (MEA1060-Inv-BC, Multi Channel Systems MCS GmbH, Germany). For long duration recording, micro incubation chamber was constructed around the MEA system with temperature controller and CO2 supply. Pharmacological experiments were performed on mature samples (> 20 days in vitro) which showed stable array-wide synchronized burst events. All the drugs used in this study were purchased from Tocris Bioscience, United Kingdom. After the pharmacological experiments, few samples were fixed with 4% PFA for immunocytochemistry. Neuronal soma and dendrites were identified with microtubule- associated protein 2 (MAP2) primary antibody and astrocytes were labeled with Glial fibrillary acidic protein (GFAP) primary antibody. All cell nuclei were stained with DAPI. Fluorescence from the immuno-labeled cultures were imaged at 10X using inverted confocal microscope (Zeiss LSM 880 system, Germany). To increase reproducibility of drug experiments, the protocol from (5) was followed. Briefly, before every experiment a sample was washed with conditioned media three times. The sample was left to stabilize for the next 15 minutes followed by baseline activity recording for reference. Test drugs were then added and incubated with continuous recording. After recording the drug effects, the sample was washed again with the same conditioned media and its recovery was recorded similarly. Results Cortical cells growing on MEA develop into randomly organized neuroglial network. Like neurons, astrocytes develop their own astrocytic network blending with their neighboring neuronal network (Fig.1C). During their development, neuronal networks exhibit rich repertoire of bursting patterns (6). By the end of third week after plating, the networks become mature exhibiting more stable bursting patterns. At this stage, usually two types of firing activities are observed; spontaneous isolated spiking activities in few electrodes or array-wide synchronized bursts (Fig.1D). The onset of typical spontaneous NS is marked by rapid recruitment of several neurons firing simultaneously and the collective firing lasts between 0.3 - 3 sec long (Fig. 1E). NS then terminate with a quiescent phase within which no neuronal activity can be observed. After this quiescent phase the network returns back to its basal firing rate until the next NS occurs. A sample reference recording before addition of test drug shows typical spontaneous network responses (Fig. 2A). On adding low concentration (100 µM) of dihydrokainic acid (DHK), a selective astrocytic GLT-1 inhibitor, the isolated spiking activities in several electrodes become evident along with fall of NS frequency (Fig. 2B). The fall of NS frequency exhibited drug dosage dependency. At 200 µM of DHK, most of the networks displayed complete abolishment of NS along with appearance of tonic spiking activity in all the recording units (Fig. 2C). After washing, all the samples displayed NS recovery and reduction in isolated spiking similar to the reference (Fig. 2D). Discussion Astrocytic GLT-1 glutamate transporters are known for maintaining neurotransmitter homeostasis and avoiding excitotoxicity by clearing released glutamate at synapses (7). Pharmacological treatment of cortical networks with GLT-1 blocker drives network into random firing state abolishing network synchrony. Removing this blocker effect, reestablishes network synchrony. The putative mechanism behind this effect could be GLT-1 blockage results in increase of unbound synaptic glutamate concentration at synapses. This in turn may result in recurrent and random sensitization of the post-synaptic neurons manifesting as increased isolated firing activities which eventually becomes tonic. In principle, this random activation of neurons should essentially enhance network synchrony. However, the observed results lead us to a hypothesis that astrocytic glutamate transporters not only maintain neurotransmitter homeostasis at synapses, they may also be messengers for network synchrony. As indicated, astrocytic uptake of glutamate can also shape kinetics of glutamatergic synaptic activity (8) via release of gliotransmitters, blocking the glutamate uptake may hinder release of astrocyte-neuron signaling molecules that result in network synchrony. Conclusion Astrocyte glutamate transporters are important for the maintenance of synchronized bursting events. These events in neuronal ensembles may be mediated through astrocytic GLT-1 glutamate transporters.

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References

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Keywords: network spike, cortical network, astrocyte, Multi-electrode arrays, glutamate recycle

Conference: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays, Reutlingen, Germany, 4 Jul - 6 Jul, 2018.

Presentation Type: Poster Presentation

Topic: Neural Networks

Citation: Kumar R, Tzeng S and Chan C (2019). Neuronal network synchronization is mediated through astrocytic glutamate transporters
. Conference Abstract: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays. doi: 10.3389/conf.fncel.2018.38.00070

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Received: 18 Mar 2018; Published Online: 17 Jan 2019.

* Correspondence: Mr. Ravi Kumar, Taiwan International Graduate Program in Interdisciplinary Neuroscience, National Cheng Kung University and Academia Sinica, Taipei, Taiwan, ravikumar@gate.sinica.edu.tw