Investigation of Seizure-Susceptibility in a Drosophila melanogaster Model of Human Epilepsy with Optogenetic Stimulation

Abstract

We examined seizure-susceptibility in a Drosophila model of human epilepsy using optogenetic stimulation of ReaChR (red-activatable channelrhodopsin). Photostimulation of the seizure-sensitive mutant para^bss1 causes behavioral paralysis that resembles paralysis caused by mechanical stimulation, in many aspects. Electrophysiology shows that photostimulation evokes abnormal seizure-like neuronal firing in para^bss1 followed by a quiescent period resembling synaptic failure and apparently responsible for paralysis. The pattern of neuronal activity concludes with seizure-like activity just prior to recovery. We tentatively identify the mushroom body as one apparent locus of optogenetic seizure initiation. The α/β lobes may be primarily responsible for mushroom body seizure induction.

Keywords: epilepsy; red light activable channelrhodopsin; seizure-suppression; sodium channel.

Figure 1

Generation of SLA in BS mutants by optogenetics. (A) Schematic diagram of stimulation and recording model of the GF circuit in Drosophila. Stimulation of the brain by electrical HFS activates the GF, which electrically synapses with the PSI. The PSI forms chemical synapses with five mns of the contralateral DLMs, also known as indirect flight muscles. (B) Bang-sensitivity seen in para^bss1 and eas BS mutants after mechanical shock. (C) Recording from a para^bss1 BS mutant DLM fiber following delivery of a 4 V HFS stimulus that is effective in evoking SLA. (D) Schematic diagram of stimulation and recording model of the GF circuit. Only the recording electrode is inserted in the DLM. (E) LS paralysis behavior observed in elav^c155para^bss1/Y;;UAS-ReaChR/+ and elav^c155 eas/Y;;UAS-ReaChR/+ double mutants. (F) Upper trace: photostimulation in control elav^c155bss/Y flies did not evoke any SLA. Lower trace: light-induced seizure showed characteristic initial seizure, synaptic failure, and recovery seizure, similar to the seizure induced by HFS. The red bar represents the time duration of photostimulation. (C and F) Horizontal calibration is 10 sec and vertical calibration is 50 mV. BS, bang-sensitive; CS, control; DLMs, dorsal longitudinal muscles; GF, giant fiber; HFS, high-frequency stimuli; LS, light sensitive; mns, motorneurons; PSI, peripherally synapsing neurons; SLA, seizure-like activity.

Figure 2

Optimization of photostimulation frequency. Maximum intensity of red light for 500 msec with 50% duty cycle of frequency range from 1 to 100 Hz was delivered to elav^c155 para^bss1/y; UAS-ReaChR/+ double mutants. There was not any significant difference in seizure-like electrical activity in double mutants at different frequency compared to continuous red light photostimulation. The red bar represents the time duration of photostimulation. In the continuous photostimulation trace, the red bar is smaller than the other frequency traces as in this case we applied continuous photostimulation with 100% duty cycle. Horizontal calibration is 10 sec and vertical calibration is 100 mV.

Figure 3

Characterization of MB and its αβ lobe as a seizure initiation site in Drosophila brain. (A) Quantification of LS paralysis behavior elicited in MB by expression of UAS-ReaChR, using different MB-GAL4 drivers. (B) Expression of UAS-ReaChR in MB by 117Y-GAL4 driver and subsequent optical stimulation by red light-elicited seizures in BS mutants. (C) Quantification of LS paralysis behavior by different MB lobe GAL4 drivers. Neither α’β’ nor γ lobe GAL4 drivers produced any LS paralysis behavior. (D) Expression of UAS-ReaChR in MB αβ lobe using 121Y-GAL4 is sufficient to trigger seizures by light stimulation. (B and D) Horizontal calibration is 10 sec and vertical calibration is 50 mV. BS, bang-sensitive; LS, light-sensitive, MB, mushroom body.