Abstract
AimGtACR2, 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.MethodsAdult 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].ResultsIn 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.ConclusionPrecisely 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.
Highlights
Optogenetics refers to a technique, where neurons are genetically modified to express non-endogenous light-sensitive proteins to reversibly modulate their activity with a high degree of spatiotemporal precision using a specific wavelength of light
In Experiment 3, we investigated the effect of activating GtACR2 on paired-pulse relations of evoked potentials (EPs), for which, paired pulses with an inter-pulse interval (IPI) of 20 ms
Illuminating the GtACR2-expressing CA1 subfield of the hippocampus with 32, 160, and 320 mW/mm2 resulted in negative field potentials at both the dorsal and ventral electrode contacts, positioned near the axons and dendrites of the CA1 neurons, respectively (Figure 1A)
Summary
Optogenetics refers to a technique, where neurons are genetically modified to express non-endogenous light-sensitive proteins (opsins) to reversibly modulate their activity with a high degree of spatiotemporal precision using a specific wavelength of light. Many in vivo studies using inhibitory opsins have resorted to inward chloride pumps (e.g., halorhodopsins; NpHR) (Sadler et al, 2017; Lee et al, 2018; Moon et al, 2018) or outward proton pumps (e.g., archaerhodopsins; ArCh) (Falgairolle and O’Donovan, 2019; Hoseini et al, 2019; Solecki et al, 2019) Efficient inhibition of neurons with these opsins requires high levels of opsin expression on the cell membrane and require higher light powers (Wietek et al, 2015) Photostimulation sensitivity of these opsins, measured as effective power density for 50% activation (EPD50), is low (NpHR and ARCh, EPD50: 5–10 mW/mm2) (Mattis et al, 2011). Applications of these opsins are further limited to brief inhibitions of neurons (typically < 15 s), as prolonged illumination with higher light powers causes heating of tissue which is an unwanted side-effect (Wiegert et al, 2017)
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