A Step-by-Step Protocol for Optogenetic Kindling

Abstract

Electrical kindling, repeated brain stimulation eventually resulting in seizures, is widely used as an animal model of epileptogenesis and epilepsy. However, the stimulation electrode used for electric kindling targets unknown neuronal populations and may introduce tissue damage and inflammation. Optogenetics can be used to circumvent these shortcomings by permitting millisecond control of activity in genetically defined neurons without gross injury or inflammation. Here we describe an easy step-by-step protocol for optogenetic kindling - optokindling - by which seizures are eventually elicited in initially healthy mice through repeated light stimulation of neurons expressing Channelrhodopsin-2 (ChR2). Chronic EEG recordings may be performed over large time scales to monitor activity while video camera monitoring may be used to assess the behavioral severity of seizures. In conclusion, with optokindling, neuroscientists can elucidate the circuit changes that underpin epilepsy while minimizing the contribution of confounding factors such as brain damage and inflammation.

Keywords: Channelrhodopsin; animal model; epilepsy; kindling; optogenetics; protocol; seizure.

FIGURE 1

Timeline of the optokindling protocol. This timeline indicates the number of days elapsed from start as well as the duration of each step. Preparation: All materials for surgery and optoelectronic components are prepared. Surgery (1 day): Animals are injected with AAVs and implanted with fiberoptic ferrules and EEG electrodes. This important step sets the foundation for the rest of the experiments. Expression (21 days): This waiting period is required to reach sufficiently high ChR2 expression levels. Habituation (3 days): To ensure that animals are not stressed by experimenter handling during the subsequent optokindling period, animals are habituated. Optokindling (50 days): While optokindling, animal behavior is monitored, and outcomes such as seizure duration and severity are quantified. The time required for optokindling is 50 days provided the 25 stimulation sessions are spaced ∼48 h apart. It is likely possible to stimulate more often, e.g., every 24 h.

FIGURE 2

Optokindling protocol for gradual increase of seizure susceptibility in vivo. (A) Coronal M1 section indicating ChR2 expression primarily in L2/3. Inset shows close-up of L2/3 ChR2-expressing PCs. (B) Bilateral implantation of recording screws allows EEG recording whilst fiber-optic ferrule implantation above pia facilitate ChR2 activation without damaging the brain. Fiber optic cables were air-coupled to 445-nm lasers. EEG signals were amplified and then digitized by a computer (not shown). (C) During each stimulation session, M1 was repeatedly exposed to 445-nm laser light (“Induction”), delivered as 15 bouts of 3-s-long 50-Hz bursts of 5-ms pulses, divided into three sweeps delivered once a minute. Sessions were repeated every 2 days, 25 times or more. In this example, a prominent seizure was evoked in the first induction sweep of session X = 15. We measured EEG responses to 30-Hz paired-pulse laser stimuli for 10 min before and 20 min after the optokindling induction to look for long-term changes in circuit plasticity. Inset: Paired-pulse EEG responses before (red) and after (blue) indicated a change in EEG dynamics but not amplitude. Reproduced from Cela et al. (2019).

FIGURE 3

Custom optokindling platform for simultaneous ChR2 excitation and EEG recording. (A) Side view A shows both laser control boxes with one 445-nm laser mounted to a breadboard. The laser air coupler collects the laser beam into an FC-PC fiber optic cable using a fiber port collimator. Top view A shows both 445-nm lasers mounted on the breadboard. (B) Side view B shows the Faraday cage where EEG recordings are performed as well as the extracellular amplifier used during recordings. (C) Side view C shows the oscilloscope used to display EEG signals during acquisition as they are digitized by the data acquisition board and stored on the computer.