The Effects of Optogenetic Activation of Astrocytes on Spike-and-Wave Discharges in Genetic Absence Epileptic Rats

Abstract

Background: Absence seizures (petit mal seizures) are characterized by a brief loss of consciousness without loss of postural tone. The disease is diagnosed by an electroencephalogram (EEG) showing spike-wave discharges (SWD) caused by hypersynchronous thalamocortical (TC) oscillations. There has been an explosion of research highlighting the role of astrocytes in supporting and modulating neuronal activity. Despite established in vitro evidence, astrocytes' influence on the TC network remains to be elucidated in vivo in the absence epilepsy (AE).

Purpose: In this study, we investigated the role of astrocytes in the generation and modulation of SWDs. We hypothesize that disturbances in astrocytes' function may affect the pathomechanism of AE.

Methods: To direct the expression of channelrhodopsin-2 (ChR2) rAAV8-GFAP-ChR2(H134R)-EYFP or to control the effect of surgical intervention, AAV-CaMKIIa-EYFP was injected into the ventrobasal nucleus (VB) of the thalamus of 18 animals. After four weeks following the injection, rats were stimulated using blue light (~473 nm) and, simultaneously, the electrophysiological activity of the frontal cortical neurons was recorded for three consecutive days. The animals were then perfused, and the brain tissue was analyzed by confocal microscopy.

Results: A significant increase in the duration of SWD without affecting the number of SWD in genetic absence epileptic rats from Strasbourg (GAERS) compared to control injections was observed. The duration of the SWD was increased from 12.50 ± 4.41 s to 17.44 ± 6.07 following optogenetic stimulation in GAERS. The excitation of the astrocytes in Wistar Albino Glaxo Rijswijk (WAG-Rij) did not change the duration of SWD; however, stimulation resulted in a significant increase in the number of SWD from 18.52 ± 11.46 bursts/30 min to 30.17 ± 18.43 bursts/30 min. Whereas in control injection, the duration and the number of SWDs were similar at pre- and poststimulus. Both the background and poststimulus average firing rates of the SWD in WAG-Rij were significantly higher than the firing recorded in GAERS.

Conclusion: These findings suggest that VB astrocytes play a role in modulating the SWD generation in both rat models with distinct mechanisms and can present an essential target for the possible therapeutic approach for AE.

Keywords: GAERS; Optogenetics; Spike-and-wave discharges; Typical absence epilepsy; WAG-Rij.

Figure 1.. Selective expression of ChR2 in (A) astrocytes and (B) not in neurons.

Figure 2.. Injection and recording of GAERS animals. (A) Virus injection sites of the VB thalamus. (B) A sample recording of a burst of SWD. (C) Representation of the optogenetic stimulation pulses on the upper part. The lower part shows a sample figure depicting the electrophysiological recording. (D) The average duration of SWD before and after optogenetic stimulation in ChR2 (left) and control (right) virus injected GAERS. (E) The average number of SWD before and after optogenetic stimulation in ChR2 (left) and control (right) virus injected GAERS. **P < .01, Error bars are standard deviation. The background and poststimulus region were 30 min long. Scale bars of the images are 400 µm for upper left, 200 µm for upper right and below left, and 100 µm for lower right.

Figure 3.. Injection and recording of WAG-Rij animals. (A) Virus injection sites of the VB thalamus. (B) A sample recording of a burst of SWD. (C) Representation of the optogenetic stimulation pulses on the upper part. The lower part shows a sample figure depicting the instantaneous firing rate of the SWD (red dots in the upper trace) and electrophysiological recording (lower trace). (D) The average duration of SWD before and after optogenetic stimulation in ChR2 (left) and control (right) virus injected WAG-Rij. (E) The average number of SWD before and after optogenetic stimulation in ChR2 (left) and control (right) virus injected WAG-Rij. **P < .01, Error bars are standard deviation. The background and poststimulus region were 30 min. Scale bars of the images are 300 µm for upper left, 400 µm for upper right and below left, and 200 µm for the lower right.

Figure 4. . The difference between background and poststimulus SWD discharge rates in (A) GAERS and (B) WAG-Rij rats. The comparison of the (C) background and (D) poststimulus firing rates of SWD between GAERS and WAG-Rij. ****P < .0001. Error bars are standard deviation.

Figure 5.. Histograms (5 s of bin width) showing the distribution of SWD duration at (A) background and (B) poststimulus region in GAERS. Histograms (1 s bin width) showing the distribution of SWD duration in WAG-Rij at (C) background and (D) poststimulus region.