Exploring for the Glymphatic System in cultured neuron-glia networks: Mapping the effect of norepinephrine on network dynamics
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1
Tel Aviv University, School of Physics and Astronomy, Israel
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2
Tel Aviv University, Faculty of Life Sciences, Israel
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3
Tel Aviv University, School of Electrical Engineering, Israel
Exploring for the Glymphatic System in cultured neuron-glia networks: Mapping the effect of norepinephrine on network dynamics
Yasmin Bar El1,*, Sivan Kanner2, Ari Barzilai2,4 and Yael Hanein3,4; 1School of Physics and Astronomy, Tel-Aviv University, Israel; 2Faculty of Life Sciences, Tel-Aviv University, Israel; 3School of Electrical Engineering, Tel-Aviv University, Israel; 4Sagol School of Neuroscience, Tel-Aviv University, Israel
*Presenting Author’s Email Address: yasminb7@mail.tau.ac.il
Keywords: Neuro-glia networks, calcium imaging, MEA recording, norepinephrine, glymphatic system.
Motivation: Recent studies showed that during sleep both the interstitial space and the flux of interstitial fluid increase as a result of glia cells shrinkage. It is suggested that the brain goes through a clearance process (named "the Glymphatic system") in which the body can remove potentially neurotoxic waste that accumulate in the central nervous system of awake animals [1]. Dysfunctionality of this process may have a key role in the development of brain degenerative diseases such as Alzheimer's disease which are typified by the accumulation of protein plaque. Independently, recent studies established the important role of astrocytes in information processing. Owing to the strong link between neurons and glia activity and morphology, it is clear that morphological changes in glia-network should be manifested in changes in glial and neuronal activities. The aim of this study is to explore these changes through careful investigation of cultured neuron-glia networks [2].
Material and Methods: In order to explore the role of astrocytes in sleep-wake cycles and specifically in the glymphatic system, we study the effect of norepinephrine (NE) on the dynamics of dissociated cultured cortical neuron-glial networks. NE is a main distinguishing factor between an awake and a sleep states and affects the activity of both neurons and glia [4]. Dissociated cortical neuro-glia cultures (SV129-mice, post-natal 0-1), were cultured on MEA (200/30iR-Ti) coated with PDL. MEA recordings (sample rate: 20 KHz) was performed simultaneously with calcium imaging microscopy of neuron and glia intracellular calcium signals (sample rate: 52Hz). This unique setup [2, 3] allows mapping of both neuronal action potentials and glia calcium waves. The MEA was also used to deliver electrical stimulation to neurons (Bi-phasic pulses, positive followed by negative, with each phase lasting 400 ?s at 20Hz, ~1.5 mC/cm2). Under these conditions we introduce norepinephrine. Immunostaining was used to map morphological changes in astrocytes in response to NE applications (50uM NE was added manually).
Results: 14 DIV neuro-glia cultures were fixated and stained with GFAP after exposure to NE. Morphological changes in astrocytes are clearly apparent (Figure 1): Control cultures are characterized by highly fibrous and entangled morphology, resembling the protoplasmic astrocytes, while that of the test cultures is uniform and spherical without apparent fibers. We hypothesized that these morphological changes may influence networks activity and accordingly explored for activity changes in astrocytes and neurons. Indeed, calcium imaging traces show marked changes in glial cells activity (Figure 2a). Before NE addition, astrocyte activity was sparse and sporadic. As seen in the calcium imaging data, after NE addition, astrocyte activity increased and was typified by clear calcium waves. Further addition of NE appears to also affect coupling between astrocytes (Figure 2b). Moreover, the Introduction of NE alters the glia response to electrical stimulation as indicated by calcium traces (Figure 2d). MEA recordings show differences in neuron bursts pattern before and after NE addition, and return to control pattern after 170 s (Figure 3).
Discussion: Our results show a first in-vitro evidence for NE effect on astrocyte morphology and network dynamics. The combined MEA and calcium imaging technique proves to be a powerful tool to perform an investigation into astrocyte-neuron communication under varying morphological changes, to explore the robustness of the electrical activity under changing parameters and possibly for assessing the efficacy of different pharmacologies under these parameters.
Fig 1: Morphological changes of astrocytes labeled with GFAP (green) in the presence of 50uM norepinephrine after 40 minutes. (a) Control conditions, (b) norepinephrine.
Fig 2: (a) Calcium traces of neurons (red) and glia (green) before and after NE (in blue). Glia 1-5 show a clear increase in activity after NE addition. (b) Traces before and after second addition. Glia 6-7 are synchronized before and go out of phase after NE second addition. (c) Fluorescence imaging on a MEA. Electrodes and Glia 6 and 7 are circumscribed. (d) Calcium traces of neurons (red) and glia (green) in the presence of electrical stimulation (gray) of 20 Hz, 25uA, before and after addition of NE (blue). Glia traces show different behavior under the electrical stimulation before and after NE addition.
Fig 3: (a) bursts before and after first addition of NE. Bursts pattern changed after NE addition (depicted with pink line) and returns to control pattern after 170 seconds. (b) 3 selected bursts of the same electrode, before (blue), right after (black) and 170 second after (red) showing the change in burst pattern.
References:
[1] Lulu Xie, Hongyi Kang, Qiwu Xu, Michael J. Chen, Yonghong Liao, Meenakshisundaram Thiyagarajan, John O’Donnell, Daniel J. Christensen, Charles Nicholson, Jeffrey J. Iliff, Takahiro Takano, Rashid Deane, Maiken Nedergaard (2013) Sleep Drives Metabolite Clearance from the Adult Brain. SCIENCE 342: 373-377.
[2] Gilad Wallach, J Lallouette, Nitzan Herzog, Maurizio De Pitta`, Eshel Ben Jacob, B Hugues, Yael Hanein. (2014) Glutamate Mediated Astrocytic Filtering of Neuronal Activity. PLoS Comput Biol 10(12): e1003964.
[3] Nitzan Herzog, Mark Shein-Idelson and Yael Hanein. (2011) Optical validation of in vitro extra-cellular neuronal recordings. IOP Publishing Ltd. Journal of Neural Engineering, Volume 8.
[4] John O'Donnell, Douglas Zeppenfeld, Evan McConnell, Salvador Pena, Maiken Nedergaard. (2012) Norepinephrine: A Neuromodulator that boosts the function of multiple cell types to optimize CNS performance. Neurochem Res (2012) 37:2496-2512.
Keywords:
Norepinephrine,
calcium imaging,
Glymphatic system,
MEA recording,
Neuro-glia networks
Conference:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays, Reutlingen, Germany, 28 Jun - 1 Jul, 2016.
Presentation Type:
oral
Topic:
MEA Meeting 2016
Citation:
Bar El
Y,
Kanner
S,
Hanein
Y and
Barzilai
A
(2016). Exploring for the Glymphatic System in cultured neuron-glia networks: Mapping the effect of norepinephrine on network dynamics.
Front. Neurosci.
Conference Abstract:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays.
doi: 10.3389/conf.fnins.2016.93.00049
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Received:
22 Jun 2016;
Published Online:
24 Jun 2016.
*
Correspondence:
Dr. Yasmin Bar El, Tel Aviv University, School of Physics and Astronomy, Tel Aviv, Israel, yasminb7@mail.tau.ac.il