Channelrhodopsin variants engage distinct patterns of network activity

Channelrhodopsins (ChRs) are light-gated ion channels that enable cell type-specific activation of neurons or neural circuits. Channelrhodopsin-2 has been widely used as a tool to probe circuit function in vitro and in vivo. Several recently developed ChR variants are characterized by faster kinetics and reduced desensitization. However, little is known about how their varying properties may regulate their interaction with local network dynamics. We compared ChR-evoked patterns of multi-unit activity and local field potentials in primary visual cortex of mice expressing three ChR variants with distinct temporal profiles: Chronos, Chrimson, and ChR2. We assessed overall activation of by measuring the amplitude and temporal progression of evoked spiking. Using gamma-range (30-80Hz) LFP power as an assay for local network engagement, we examined the recruitment of cortical network activity by each tool. All ChR variants caused light-evoked increases in firing in vivo, but each demonstrated different temporal patterning of evoked activity. In addition, the three ChRs had distinct effects on cortical gamma-band activity. Our findings suggest that variations in the kinetics of optogenetic tools can substantially affect their efficacy in neural networks in vivo, as well as the manner in which their activation engages circuit resonance.


Introduction
The advent of easily accessible optogenetic tools for manipulating neural activity has substantially altered experimental neuroscience. The current optogenetics toolkit for neuroscience comprises a large number of Channelrhodopsins (ChRs), Halorhodopsins, and Archaerhodopsins that enable activation and suppression of neural activity with millisecond-timescale precision. Within the Channelrhodopsin family, many variants have now been made with altered activation spectra, photocycle kinetics, and ion selectivity.
The first tool to be widely used in neuroscientific approaches, Channelrhodopsin-2 (ChR2), is a nonspecific cation channel with sensitivity to blue light.
ChR2 conferred the ability to evoke action potentials with high precision and reliability across a wide range of cell types. 1-3 However, the utility of this tool has been somewhat limited by its relatively long offset kinetics and fairly rapid inactivation of photocurrents in response to sustained strong light stimulation. 1, [4][5][6] In addition, most naturally occurring Channelrhodopsins are sensitive to blue-green light, presenting a challenge to the use of multiple tools for simultaneous optogenetic control of distinct neural populations. A significant effort in the field has therefore been made to develop Channelrhodopsin variants with faster on-and offset temporal kinetics, less desensitization over time, and red-shifted wavelength sensitivity.
Previous work has suggested that the Channelrhodopsins are highly effective tools for probing the cellular interactions underlying intrinsically generated patterns of brain activity. Stimulation of Parvalbumin (PV)-expressing interneurons in the cortex via ChR2 evokes gamma oscillations, entrains the firing of excitatory pyramidal neurons, and regulates sensory responses. 2,7 Similarly, ChR2 stimulation of PV + GABAergic long-range projection neurons in the basal forebrain generates gamma-range oscillations in frontal cortex circuits. 8 Recent work further suggests that ChR2 activation of Somatostatin-expressing interneurons, which synapse on both PV + cells and excitatory neurons, evokes cortical oscillations in a low gamma range. 9 Sustained depolarization of excitatory sensory cortical neurons via ChR2 activation likewise evokes gamma oscillations, likely by engaging reciprocal interactions with local GABAergic interneurons. 10 In comparison, activation of pyramidal neurons in mouse motor cortex via ChRGR, another ChR variant, evokes activity in a broad range of lower-band frequencies. 11 High-fidelity spiking recruited by Chronos, oChiEF, and ReaChR has been used in vitro and in vivo in visual cortex [12][13][14] and the auditory midbrain 15,16 , but the impact of such stimulation on the surrounding network remains unclear.
Despite the substantial increase in available ChR variants with diverse kinetic and spectral properties, it remains unclear how these properties interact with endogenous temporal patterns of neural circuit activity like gamma oscillations in vivo.
Furthermore, the properties of optogenetic tools are typically tested using short pulses (1 to 100ms) under quiet conditions in vitro, but these tools are widely used for sustained neural activation (100s of ms to s) under active network conditions in vivo. Here we tested the impact of optogenetic tool properties on evoked activity patterns in the intact brain. We took advantage of the well-characterized gamma oscillation rhythm in mouse primary visual cortex in vivo 10,17,18 as a metric for optogenetic recruitment of local network activity. Using optogenetic activation of excitatory pyramidal cells as a paradigm to evoke both spiking and cortical gamma oscillations, we compared three Channelrhodopsins with robust photocurrents but distinct kinetic profiles: Chronos, with high-speed on and off kinetics 19 ; ChR2, with fast on but relatively slow off kinetics 1 ; and Chrimson 19 , with slow on and off kinetics. We found that these tools, although expressed in the same cell type in the same brain region and effective at eliciting action potentials, evoked distinct patterns of activity and had different effects on gamma rhythms. Together, our data suggest that the kinetic properties of engineered opsin tools affect optogenetic interactions with local circuit activity and should be a key factor in experimental design.

Animals
All animal handling and maintenance was performed according to the regulations of the Institutional Animal Care and Use Committee of the Yale University School of Medicine. We used both female and male C57BL/6J mice ranging from 3-5 months old.

Surgical procedures
To express ChR2, Chronos, and Chrimson in pyramidal neurons, we injected

Electrophysiological recordings
Mice were anesthetized with 0.3-0.5% isoflurane in oxygen and head-fixed by cementing a titanium headpost to the skull with Metabond (Butler Schein). All scalp incisions were infused with lidocaine. A craniotomy was made over primary visual cortex and electrodes were lowered through the dura into the cortex. All extracellular multi-unit and LFP recordings were made with an array of independently moveable tetrodes mounted in an Eckhorn Microdrive (Thomas Recording). Signals were digitized and recorded by a Digital Lynx system (Neuralynx). All data were sampled at 40kHz. All LFP recordings were referenced to the surface of the cortex. LFP data were recorded with open filters and MU data were recorded with filters set at 600-9000Hz.
Optogenetic stimulation was provided via an optical fiber (200um) coupled to a laser (Optoengine) at either 470nm (ChR2 and Chronos stimulation) or 593nm (Chrimson stimulation). In each experiment, the fiber was placed on the surface of the dura over the virus injection site and the tetrodes were placed immediately posterior to the fiber.
During each experiment, a total of 150 laser pulses (470 or 593nm) of 1.5s duration were given at varying light intensities (0.5-10mW/mm 2 ) with 10s inter-pulse intervals. Bouts of 30 pulses were separated by 5-minute baseline periods.

Histology
Mice were perfused with 0.1M PBS followed by 4% PFA in 0.1M PBS. After perfusion, brains were postfixed for 8 hours in 4% PFA. Brains were sliced at 40µm on a vibratome (Leica) and mounted on slides with DAPI mounting solution (Vector). Images were taken at 10x on an Olympus microscope and the channels were merged using ImageJ (NIH). Laminar distribution of opsin expression was estimated based on DAPI staining.

Data Analysis
Data were analyzed using custom scripts written in Matlab (The Mathworks) and Igor Pro (Wavemetrics). Spikes were detected from the MU recordings using a threshold of +3 SD above the mean, where both the mean and SD were calculated from 10 seconds of recording preceding any light stimulation. Detected spikes were then used to calculate peristimulus time histogram (PSTH) and raster plots for visualization of optically evoked spiking. All firing rate measurements were normalized to the firing rate during a 10-second period prior to all light stimulations. Paired measurements were then taken for the pre-stimulus baseline period prior to each light pulse and the first 1 second of the light-evoked response to that light pulse. For each light intensity, a two-tailed unpaired t-test was performed on the normalized firing rates in the baseline and evoked conditions to determine the presence or absence of an evoked change in firing rate.
Inter-spike Intervals (ISI) were calculated as the time interval between successive spikes and a cumulative distribution of ISIs in the on-pulse and off-pulse periods was calculated for each data set Spectrograms of LFP activity were obtained using 400ms-long Hann windows sliding by 10ms. Prior to STFT, the mean was subtracted to remove DC bias.

Statistics
For most comparisons, a two-tailed t-test was used. In cases where nonparametric statistics were appropriate due to non-normal data distributions, a twotailed Kolmogorov-Smirnov test was used.

Cell type-specific expression of Channelrhodopsins in mouse visual cortex
To understand the efficacy and utility of recently developed Channelrhodopsin variants with differing kinetic properties, we compared three tools: Channelrhodopsin-2, Chronos, and Chrimson (Fig. 1a). We expressed each tool using an AAV construct, under the control of the excitatory neuron-specific CaMKII promoter, into the visual cortex of wild-type mice. Four weeks after virus injection, each of the three Channelrhodopsins was robustly expressed in a characteristic distribution of excitatory pyramidal neurons in cortical layers 2/3, 5, and 6 ( Fig. 1c). 2,20 In each case, opsin expression was widespread in visual cortex, covering up to a distance of up to about 410 µm from the initial injection site. (Fig.1b)

Different Channelrhodopsins evoke distinct cortical activity profiles in vivo
The temporal profile of circuit activity evoked by different opsins may differentially engage network dynamics. To assess the initial and sustained levels of spiking evoked by each opsin, we recorded population multiunit (MU) and local field potential (LFP) activity at multiple cortical sites around each viral injection (ChR2: 11 sites in 3 animals, Chronos: 5 sites in 3 animals, Chrimson: 11 sites in 3 animals). When stimulated with 1.5 seconds of continuous light in an appropriate wavelength (10mW/mm2, Wavelength: 470nm for ChR2 and Chronos, 593nm for Chrimson), all three ChRs evoked sustained firing ( Fig. 2a-c). However, each opsin was associated with a distinct temporal profile of spiking. Whereas stimulation of ChR2- (Fig. 2a,d) or Chrimson- (Fig. 2c,f) expressing neurons evoked sustained firing over ~1-2s, stimulation of Chronos-expressing neurons generated strong initial spiking followed by a decrease towards baseline firing levels (Fig.   2b,e). In contrast, the peak firing evoked by ChR2 and Chronos was rapid and reliable, whereas the peak firing achieved by Chrimson stimulation was delayed and highly variable ( Fig. 2d-f inset panels).
To quantify these differences in temporal kinetics induced by the three ChR variants, we compared the time between the light pulse onset and the center of the 10 ms interval with the most frequent spikes, averaged over all recording sites and mice for each ChR variant. Chronos showed the shortest peak latency of 0.005 ± 0.01s, whereas Chrimson had a peak latency of 0.43 ± 0.10s, compared to ChR2 0.014 ± 0.01s. (Fig.   2d-f, ChR2: n=3, 11 sites, Chronos: n=3, 5 sites, Chrimson: n=3, 11 sites). The latency to peak was thus shorter for Chronos (p < 0.001) and longer for Chrimson (p < 0.001) compared to ChR2 (Unpaired t-test).
To assay the efficacy of each optogenetic tool in engaging local cortical neurons, we compared the recruitment of spikes in response to a range of illumination intensities.
We examined the difference in spike frequency between the baseline and stimulation periods by comparing the distributions of inter-spike intervals (ISIs) evoked by activation of ChR2, Chronos, and Chrimson (Fig. 3). ChR2 (Fig. 3a) and Chrimson (Fig. 3c) both evoked a robust decrease in ISI, consistent with the sustained increase in firing rate, whereas activation of Chronos (Fig 3b) had only a modest effect on the overall ISI distribution. ChR2- (Fig. 3d) and Chrimson- (Fig. 3f)

Opsin-specific recruitment of cortical gamma rhythms
Previous work has found that ChR2 stimulation of pyramidal neurons engages the cortical gamma rhythm (30-80Hz), an outcome of resonant excitatory-inhibitory circuit interactions 21 , in vitro and in vivo. 10 Using evoked gamma power as a measure of network activation, we assayed the efficacy of each optogenetic tool in driving recurrent circuit interactions. Activation of ChR2-expressing excitatory neurons evoked a response in the local field potential (LFP) and a broadband increase in high-frequency activity (Fig.   4a, Sup Fig.2d). Chronos and Chrimson likewise evoked an initial deflection of the LFP signal ( Fig. 4b-c; Sup Fig. 2b,c). However, neither Chronos nor Chrimson activation of excitatory neurons evoked the characteristic sustained high-frequency LFP activity observed following ChR2 stimulation of the same population of neurons.
In agreement with previous work 10  Recently developed Channelrhodopsins vary extensively in their kinetic profiles.
ChR2 exhibits a relatively fast onset (τ on ) but a long offset time (τ off ), leading to diminished temporal fidelity in spike responses. 22 The τ off of several ChRs also slows further upon membrane depolarization. 23 The cumulative effect of this long τ off is to cause a prolonged depolarization after the evoked action potential, preventing rapid rehyperpolarization of the membrane and contributing to artificial spike doublets.
Prolonged depolarization may also inactivate voltage-gated channels needed for highfrequency spiking. Mutations in ChR2 to accelerate the closure of the pore gave rise to the CheTA variant, which has high temporal fidelity but reduced light sensitivity and charge transfer. 22 ChRGR, a ChR1 variant, shows rapid τ on and τ off , along with reduced desensitization. 11 In comparison, Chronos and ChIEF exhibit large photocurrents, rapid deactivation, and improved efficacy in eliciting high-fidelity fast spiking. 19,24 A second series of tools were developed with absorption spectra shifted towards longer wavelengths compatible with 2-photon imaging [25][26][27] and dual-channel optogenetic circuit interrogation. 19 Initial chimerization between VChR1 and ChR1 gave the redshifted C1V1 28 , with a red-shifted absorption peak but relatively low photocurrents and very slow kinetics. Further work produced several additional red-shifted tools, including ReaChR 29 , with a peak similar to C1V1 but larger photocurrents, and bReaCHES 30 , a faster variant with larger photocurrents and higher spike fidelity. In comparison, Chrimson exhibits a more red-shifted absorption peak and very large photocurrents, making it highly effective for driving robust neural activity. 19 Chrimson has substantially slower τ on and τ off properties than ReaChR, bReaCHES, ChR2, or Chronos. Based on their low toxicity, robust expression levels, large peak photocurrents, and distinct kinetic profiles, we selected Chronos and Chrimson for in vivo comparison with ChR2.
We found a strong relationship between the properties of the individual opsins and the temporal profile of the spiking they evoked. The two tools with relatively rapid onset kinetics, ChR2 and Chronos, each evoked a precisely timed initial spike event across the neuronal population, followed by sustained spiking at lower firing rates. In contrast, Chrimson, with slow onset kinetics, did not evoke reliable spiking at stimulation onset and gave rise to a much broader temporal distribution of spike frequencies. These results, along with previous findings 6,23 , suggest that the kinetics of the opsins interact meaningfully with intrinsic neuronal membrane properties. Rapid membrane depolarization, like that caused by ChR2 or Chronos activation, contributes to recruitment of voltage-gated channels and enhances the reliability and precision of the initial evoked action potentials in cortical neurons. 31,32 In comparison, a slow rate of depolarization, like that caused by Chrimson, leads to temporally disbursed spiking. We further found that the kinetics of the opsins shaped the overall temporal envelope of the sustained spiking evoked by long stimulation. Whereas the initial efficiacy of ChR2 and Chronos resulted in an early peak in evoked firing rates within the first 50ms, the spike response to Chrimson stimulation peaked several hundred milliseconds later. However, the sustained firing rates evoked by ChR2 and Chrimson were higher than that evoked by Chronos, suggesting that rapid deactivation of this opsin may reduce overall spike rates.
Gamma oscillations are generated by rhythmic interactions between excitatory and inhibitory neurons. 21 Activation of excitatory neuron synaptic input to predominantly fast-spiking interneurons causes a highly synchronous and precise spike response in the interneurons, temporarily suppressing excitatory neuron spiking. When excitatory spiking recovers following the inhibitory event, the interneurons are again recruited. This temporally structured, reciprocal interaction between excitation (E) and inhibition (I) leads to a very robust 30-80Hz network oscillation with a ~25ms period determined by the time course of inhibition. Gamma activity in cortical circuits can be evoked by optogenetically stimulating either the inhibitory interneurons 2,7,9 or the excitatory neurons. 10,33 Generation of gamma oscillations by excitatory neuron stimulation likely results from the highly synchronous activation of a large volley of spikes from excitatory neurons, which are highly effective in activating the inhibitory neuron spiking that sets the temporal pattern for resonance in the network. 2,7,21,34 Several cycles of gamma can be produced by even a single brief stimulation of excitatory pyramidal neurons 7 , but sustained gamma oscillations in active cortical networks in vivo may require consistently elevated excitatory spiking. 34,35 Given these temporal constraints, the different temporal profiles of evoked excitatory neuron spiking evoked by the three ChRs could potentially engage varying network responses.
In good agreement with previous work 10 , we found that ChR2 stimulation of excitatory pyramidal neurons evokes robust cortical gamma activity. In contrast, stimulation of the same neuronal population via Chronos evoked little to no gamma activity, presumably because the initial, highly precise spiking from stimulated cells is not followed by sufficiently elevated excitatory spiking to sustain network oscillations. In comparison, Chrimson might be expected to evoke little gamma because the slow increase in activity precludes an initial burst of spikes. Suprisingly, we found that stimulation via Chrimson also significantly suppressed endogenous gamma. These results suggest that Chrimson's slow temporal kinetics and late firing peak destabilize the highly precise interplay between E and I cells, increasing the firing rates of excitatory neurons but precluding their entrainment by inhibition.
Overall, we found that differences in the properties of three Channelrhodopsins