Synaptic densities in hypoxia exposed and electrically stimulated primary neural cultures
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1
University of Twente, Clinical Neurophysiology, Netherlands
Motivation
Prolonged ischemia leads to necrosis of neurons and glial cells, but limited periods (after cardiac arrest) or partial ischemia e.g. in the area surrounding the core of a brain infarct (penumbra), allow cell survival. Under these conditions, massive synaptic failure strongly reduces neural activity. Activity may eventually recover or further deteriorate toward massive cell death. Neurons need activity to survive (Ghosh, Carnahan, & Greenberg, 1994; Mao, Bonni, Xia, Nadal-Vicens, & Greenberg, 1999), and we hypothesize that the lack of activity may contribute to progression towards cell death. Earlier work of our group indicated that hypoxia reduces the synaptic density, and that pharmacological stimulation after hypoxia, results in increased synapse density (Stoyanova, Hofmeijer, van Putten, & Le Feber, 2015). Here, we investigated the effect of electrical stimulation on recovery and synapse density in cortical cultures.
Methods
Cells were obtained from brain cortices of newborn Wistar rats on the day of the birth and plated on a micro electrode array (MEA) precoated with poly ethylene imine (PEI). MEAs were placed under a Plexiglas hood, and a humidified gas mixture of air and N2 supplemented with 5% CO2, was blown over the setup at a rate of 2 l/min. Mixtures of air and N2 could be delivered at any ratio and were computer controlled by mass flow controllers. Normoxic conditions were realized by setting the flow controllers to 100% air; hypoxic mixtures contained 10% air and 90% N2.
First, we determined the effect of electrical stimulation during hypoxia (pO2 ≈ 20 mmHg) on post hypoxia recovery. At the beginning of each experiment, 2–3 electrodes were selected for stimulation that induced clear network responses. Array wide firing rates were determined before (6h baseline), during (24h), and after hypoxia. Cultures were stimulated during the first ten minutes of every hour (biphasic current pulses, (200 μs per phase, 12–32 μA); Inter pulse intervals: 5-10s). Control cultures were subjected to the same protocol, but not stimulated.
To investigate the effect of stimulation on synaptic density, other cultures, exposed to the same hypoxic conditions, were stimulated for 6h, starting 3h after reoxygenation. All electrodes were randomly stimulated with an interstimulus interval of 2.5 seconds. Then, cultures were fixed in paraformaldehyde and stained for the synaptic vesicle transmembrane protein synaptophysin G (Abcam, Cambridge,UK, dilution 1:1000) using avidin-biotin-horseradish peroxidase (ABC 1:500; Vector Labs, Burlingame, CA, USA). Microscopic images of neuronal somata were obtained at 60x. The immunoreactivity was readily discernible at the light microscopic level by the presence of dark-gray granules of immunoreactive product. Synapses on top of the neural somata were counted and the area was determined (Fig 2A) to calculate synaptic density. Control cultures were not stimulated and fixed 9h after reoxygenation. Both groups were compared to cultures that were not exposed to hypoxia.
Images were analyzed using a partly automated counting algorithm that was designed in Matlab which was validated against manual counting (Fig 2B; marker ‘*’). Images were filtered with a two-dimensional moving average filter (order: 9), and local maxima in blackness of the images that exceeded a set threshold were counted as synapses (Fig 2B; marker ‘0’). Sensitivity to small changes in threshold setting were evaluated by repeating the analysis using thresholds that were 5% higher (marker ‘-‘) or lower marker ‘+’).
Results
To determine the effect of stimulation during hypoxia, seven cultures were stimulated, while five control cultures were not stimulated. Although stimulus responses largely decreased during hypoxia (between red lines), electrical stimulation was able to increase from 0.08 to 0.20 (t-test: p=1*10^-7, errorbars indicate SEM differences between cultures) activity during hypoxia (Fig 1). Moreover, post hypoxia activity of stimulated cultures (blue) was substantially higher than in control cultures (green).
To determine how hypoxia affected the synaptic density, one culture was fixed after 24h of hypoxia another after a matched period of normoxic incubation. Samples were stained and photographed to estimated synaptic densities. Manual and automatic counting showed good similarity (correlation coefficient (R^2) of 0.76). On average, synaptic densities were ~20% lower in cultures that were subjected to hypoxic conditions than cultures incubated under normoxic conditions (t-test: p=0.03 ‘*’). Small threshold adjustments affected the absolute numbers of synapses counted in all conditions, but did not influence the observed decrease (23.2% vs 23.2% vs 21.0%, see Fig 2C). Neurons that received electrical stimulation during the hypoxic period showed a similar (p=0.14) synaptic density as neurons only subjected to hypoxic conditions.
Discussion
We showed that, without stimulation, the synaptic density decreases after a period of hypoxia, in agreement with earlier work (Stoyanova 2015). This might account for the decreasing activity and eventually lead to neuronal death. Electrical stimulation during hypoxic conditions is able to increase neural activity during hypoxia and post hypoxia survival of cells. It is not clear yet how the activity increase relates to changes in synaptic density. In the current data set post-hypoxic electrical stimulation was unable to restore the initial synaptic density. However, this set was limited, mainly due to staining problems in several of the fixed cultures. Future work will be aimed at expanding this set. If successful, we will also investigate the effect of electrical stimulation during hypoxia on synaptic density.
References
Ghosh, A., Carnahan, J., & Greenberg, M. E. (1994). Requirement for BDNF in Activity-Dependent Survival of Cortical Neurons. Science, 263(5153), 1618–1623. https://doi.org/10.1126/science.7907431
Guilbert, J. J. (2003). The world health report 2002 - reducing risks, promoting healthy life. Education for Health (Abingdon, England), 16(2), 230. https://doi.org/10.1080/1357628031000116808
Mao, Z., Bonni, a, Xia, F., Nadal-Vicens, M., & Greenberg, M. E. (1999). Neuronal activity-dependent cell survival mediated by transcription factor MEF2. Science (New York, N.Y.), 286(5440), 785–790. https://doi.org/10.1126/science.286.5440.785
Stoyanova, I. I., Hofmeijer, J., van Putten, M. J. A. M., & Le Feber, J. (2015). Acyl Ghrelin Improves Synapse Recovery in an In Vitro Model of Postanoxic Encephalopathy. Molecular Neurobiology. https://doi.org/10.1007/s12035-015-9502-x
Keywords:
hypoxia,
Cortical cultures,
synaptic failure,
Counting algorithm,
neuronal functioning
Conference:
MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays, Reutlingen, Germany, 4 Jul - 6 Jul, 2018.
Presentation Type:
Poster Presentation
Topic:
Stimulation strategies
Citation:
Hassink
GC,
Levers
M,
Kamphuis
S,
Born
I,
Hofmeijer
J and
Le Feber
J
(2019). Synaptic densities in hypoxia exposed and electrically stimulated primary neural cultures.
Conference Abstract:
MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays.
doi: 10.3389/conf.fncel.2018.38.00057
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Received:
17 Mar 2018;
Published Online:
17 Jan 2019.
*
Correspondence:
PhD. Gerco C Hassink, University of Twente, Clinical Neurophysiology, Enschede, Netherlands, g.c.hassink@utwente.nl