Mapping direct and indirect activation of cortical neurons by weak electrical pulses with massively parallel MEA-based electrical stimulation and recording
-
1
AGH University of Science and Technology, Faculty of Physics and Applied Computer Science, Poland
-
2
University of California, Santa Cruz, Santa Cruz Institute for Particle Physics, United States
-
3
Indiana University Bloomington, Department of Physics, United States
-
4
University of Strathclyde, Institute of Photonics, United Kingdom
Motivation
The ability of multielectrode arrays to stimulate neuronal networks at multiple locations, and to simultaneously record the stimulated activity over a large area make it a great tool for the systematic study of the large-scale effects of electrical current stimulation on neural network activity. The MEA-based studies of rodent brain slices reported here provide the lowest values of threshold currents and threshold charge for efficient activation of cortical neurons that can be found in the literature. Here we used an advanced MEA-based system to study, on a large scale, the neuronal responses to electrical pulses of low current amplitudes, down to the sub-microampere range.
Material and Methods
We used a 512-electrode array with an active area of 2 x 1 mm2, a spatial resolution of 60 µm and an electrode diameter of 5 µm. The MEA was combined with custom electronics based on dedicated integrated circuits to simultaneously stimulate and record the neuronal responses from organotypic rat and mouse cortical cultures. We optimized the stimulation protocol to study the neuronal activation as a function of stimulus amplitude (0.3 - 3.9 µA, 10% amplitude steps) and spatial location (64-128 stimulating electrodes for a single preparation). The stimulus artifacts were minimized using triphasic pulse waveform [1] and blanking technique, providing efficient recording across the array as soon as 1 ms after the stimulation pulse. We identified time-locked spiking activities across the array for each stimulating electrode, and fit the PSTHs with Gaussian functions to describe the latency and jitter of each stimulation response. Only responses with good fit quality were accepted.
Results
The spatial distribution and intensity of stimulated spiking activity in the mouse preparation as a function of stimulation current, for one stimulating electrode, is shown in figure 1A. Several electrodes exhibited activity clearly time-locked to the stimulus even for currents below 1 µA, including the lowest current used for measurement (0.4 µA). Virtually all the electrodes stimulated activity in distant locations within the used range of stimulation amplitudes. On average, stimulation with 1 µA pulses initiated time-locked activity at 10 electrodes across the array (figure 1B).
In the rat preparation we identified two types of neuronal responses, as shown in figure 2 (A-D). The plot 2A shows a clear two-peak distribution of the jitter values, with the first peak centered at around 0.1 ms and the second peak centered at 1.2 ms. The clear distinction between these two classes suggest two different mechanisms of neuronal activation. Based on previously published reports [2] we hypothesize that the low-jitter responses were recorded from neurons activated directly (most likely antidromically via their axonal fibers) and the large-jitter responses were recorded from neurons that were activated indirectly, that is by signals transmitted synaptically from directly activated cells. We associated the functional connections with a jitter below 0.4 ms with direct activation, and those with a jitter above this value with indirect activation. We repeated the stimulation protocol with the same culture preparation after application of synaptic blockers DNQX and APV (50 µM and 20 µM, respectively). Consistent with the expectation, the synaptic blockers removed the large-jitter responses but left intact the low-jitter responses (figure 2 (E-H)). In total, we identified 2068 direct and 937 indirect functional connections in the rat preparation, that is, respectively, 68.8% and 31.2% of the total number of 3005 connections. In contrast, in the mouse preparation we found virtually no indirect functional connections.
With respect to the neuronal responses at very low stimulation currents, we found time-locked activity with at least a 25% response rate for the lowest stimulation currents used in both the rat and mouse experiments. In the mouse experiments, 29 out of 128 stimulating electrodes (22.7%) initiated neuronal activity at one or more recording sites at a current amplitude of 0.4 µA. In the rat experiment, 4/64 electrodes (6.3%) stimulated neuronal activity at a 0.3 µA amplitude. These are, to our knowledge, the lowest thresholds for effective extracellular stimulation of neurons in brain tissue reported so far.
Discussion and Conclusion
In this study we report for the first time on the activation of neuronal fibers in cortex by sub-microampere electrical currents (although similar or even lower thresholds were found for stimulation of neurons in retina and spinal cord). In addition, in our rat experiment, we identified both direct and indirect neuronal activation, but in the mouse recording we detected almost no indirect activation.
Among published work, the lowest reported current threshold for cortical axons was 1.0 µA (3.3 times our lowest threshold) while the lowest charge threshold was 55 pC (1.75 times our lowest threshold). We hypothesize that finding these low-amplitude responses is, at least partially, a matter of statistics. By stimulating the slices at 64-128 locations, combined with recording at 505 electrodes, we were able to identify low-probability effects of neuronal stimulation with low-current electrical pulses. We also note that several other published studies, based on electrodes of larger diameters, reported significantly lower current density thresholds than observed in our experiments.
Figure 1. Low-amplitude current stimulation in the mouse cortical preparation. (A) Number of time-locked spikes associated with the electrical stimulation applied to a single electrode (marked with the green circle) as a function of current amplitude. The current value in µA is given in the upper left-hand corner of each subplot. The colorbar indicates the number of time-locked spikes associated with each recording electrode. (B) Average number of electrodes with time-locked activity per single stimulating electrode as a function of the stimulation current.
Figure 2. Direct and indirect activation in rat preparation. (A-D): no drugs, (E-H): with synaptic blockers. Plots (A) and (E) show histogram of spike jitters for all identified functional connections. The time bin width is 50 µs. For plots (B-D) and (F-H) a single connection is marked with a single diamond. Red diamond color: low jitter responses (sigma <0.4 ms). Blue color: large-jitter responses (sigma >0.4 ms).
Acknowledgements
This work was supported by Polish National Science Centre grant 2013/08/W/NZ4/00691 (PH), National Science Foundation grant 1513779 (JMB)
References
[1] Hottowy P, Skoczen A, Gunning DE, Kachiguine S, Mathieson K, Sher A, Wiacek P, Litke AM, Dabrowski W: “Properties and application of a multichannel integrated circuit for low-artifact, patterned electrical stimulation of neural tissue”, Journal of Neural Engineering, 9, pp. e066005 (2012).
[2] Wagenaar DA, Pine J, Potter SM: “Effective parameters for stimulation of dissociated cultures using multi-electrode arrays”, Journal of Neuroscience Methods 138, pp. 27-37 (2004).
Keywords:
Electrical Stimulation,
nanostimulation,
Direct activation,
Indirect activation,
synaptic blocker
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:
Hottowy
P,
Yeh
F,
Ito
S,
Skoczen
A,
Wiacek
P,
Mathieson
K,
Dabrowski
W,
Beggs
JM and
Litke
AM
(2019). Mapping direct and indirect activation of cortical neurons by weak electrical pulses with massively parallel MEA-based electrical stimulation and recording.
Conference Abstract:
MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays.
doi: 10.3389/conf.fncel.2018.38.00024
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:
Dr. Pawel Hottowy, AGH University of Science and Technology, Faculty of Physics and Applied Computer Science, Kraków, 30059, Poland, hottowy@agh.edu.pl