In vivo inhibition of epileptiform afterdischarges in rat hippocampus by light-activated chloride channel, stGtACR2.

AIMS: The blue light-sensitive chloride-conducting opsin, stGtACR2, provides potent optogenetic silencing of neurons. The present study investigated whether activation of stGtACR2 in granule cells of the dentate gyrus (DG) inhibits epileptic afterdischarges in a rat model. METHODS: Rats were bilaterally injected with 0.9 µl of AAV2/7-CaMKIIα-stGtACR2-fusionred in the DG. Three weeks later, afterdischarges were recorded from the DG by placing an optrode at the injection site and a stimulation electrode in the perforant path (PP). Afterdischarges were evoked every 10 min by unilateral electrical stimulation of the PP (20 Hz, 10 s). During every other afterdischarge, the DG was illuminated for 5 or 30 s, first ipsilaterally and then bilaterally to the PP stimulation. The line length metric of the afterdischarges was compared between illumination conditions. RESULTS: Ipsilateral stGtACR2 activation during afterdischarges decreased the local field potential line length only during illumination and specifically at the illuminated site but did not reduce afterdischarge duration. Bilateral illumination did not terminate the afterdischarges. CONCLUSION: Optogenetic inhibition of excitatory neurons using the blue-light sensitive chloride channel stGtACR2 reduced the amplitude of electrically induced afterdischarges in the DG at the site of illumination, but this local inhibitory effect was insufficient to reduce the duration of the afterdischarge.

In vivoblue light illumination for optogenetic inhibition: effect on local temperature and excitability of the rat hippocampus.

Objective.The blue light-activated inhibitory opsin, stGtACR2, is gaining prominence as a neuromodulatory tool due its ability to shunt-inhibit neurons and is being frequently used inin vivoexperimentation. However, experiments involving stGtACR2 use longer durations of blue light pulses, which inadvertently heat up the local brain tissue and confound experimental results. Therefore, the heating effects of illumination parameters used forin vivooptogenetic inhibition must be evaluated.Approach.To assess blue light (473 nm)-induced heating of the brain, we used a computational model as well as direct temperature measurements using a fiber Bragg grating (FBG). The effects of different light power densities (LPDs) and pulse durations on evoked potentials (EP) recorded from dentate gyrus were assessed. For opsin-negative rats, LPDs between 127 and 636 mW mm-2and pulse durations between 20 and 5120 ms were tested while for stGtACR2 expressing rats, LPD of 127 mW mm-2and pulse durations between 20 and 640 ms were tested.Main results.Increasing LPDs and pulse durations logarithmically increased the peak temperature and significantly decreased the population spike (PS) amplitude and latencies of EPs. For a pulse duration of 5120 ms, the tissue temperature increased by 0.6 °C-3.4 °C. All tested LPDs decreased the PS amplitude in opsin-negative rats, but 127 mW mm-2had comparatively minimal effects and a significant effect of increasing light pulse duration was seen from 320 ms and beyond. This corresponded with an average temperature increase of 0.2 °C-1.1 °C at the recorded site. Compared to opsin-negative rats, illumination in stGtACR2-expressing rats resulted in much greater inhibition of EPs.Significance.Our study demonstrates that light-induced heating of the brain can be accurately measuredin vivousing FBG sensors. Such light-induced heating alone can affect neuronal excitability. Useful neuromodulation by the activation of stGtACR2 is still possible while minimizing thermal effects.

Attenuation of Hippocampal Evoked Potentials in vivo by Activation of GtACR2, an Optogenetic Chloride Channel.

AIM: GtACR2, a light-activated chloride channel, is an attractive tool for neural inhibition as it can shunt membrane depolarizations. In this study, we assessed the effect of activating GtACR2 on in vivo hippocampal CA1 activity evoked by Schaffer collateral (SC) stimulation. METHODS: Adult male Wistar rats were unilaterally injected with 0.5 µL of adeno associated viral vector for induction of GtACR2-mCherry (n = 10, GtACR2 group) or mCherry (n = 4, Sham group) expression in CA1 pyramidal neurons of the hippocampus. Three weeks later, evoked potentials (EPs) were recorded from the CA1 subfield placing an optrode (bipolar recording electrode attached to an optic fiber) at the injection site and a stimulation electrode targeting SCs. Effects of illumination parameters required to activate GtACR2 such as light power densities (LPDs), illumination delays, and light-pulse durations were tested on CA1 EP parameters [population spike (PS) amplitude and field excitatory postsynaptic potential (fEPSP) slope]. RESULTS: In the GtACR2 group, delivery of a 10 ms light-pulse induced a negative deflection in the local field potential which increased with increasing LPD. When combined with electrical stimulation of the SCs, light-induced activation of GtACR2 had potent inhibitory effects on CA1 EPs. An LPD of 160 mW/mm2 was sufficient to obtain maximal inhibition CA1 EPs. To quantify the duration of the inhibitory effect, a 10 ms light-pulse of 160 mW/mm2 was delivered at increasing delays before the CA1 EPs. Inhibition of EPs was found to last up to 9 ms after the cessation of the light-pulse. Increasing light-pulse durations beyond 10 ms did not result in larger inhibitory effects. CONCLUSION: Precisely timed activation of GtACR2 potently blocks evoked activity of CA1 neurons. The strength of inhibition depends on LPD, lasts up to 9 ms after a light-pulse of 10 ms, and is independent of the duration of the light-pulse given.