Investigating the feasibility of channelrhodopsin variants for nanoscale optogenetics

Abstract. Optogenetics has revolutionized the study of circuit function in the brain, by allowing activation of specific ensembles of neurons by light. However, this technique has not yet been exploited extensively at the subcellular level. Here, we test the feasibility of a focal stimulation approach using stimulated emission depletion/reversible saturable optical fluorescence transitions-like illumination, whereby switchable light-gated channels are focally activated by a laser beam of one wavelength and deactivated by an overlapping donut-shaped beam of a different wavelength, confining activation to a center focal region. This method requires that activated channelrhodopsins are inactivated by overlapping illumination of a distinct wavelength and that photocurrents are large enough to be detected at the nanoscale. In tests of current optogenetic tools, we found that ChR2 C128A/H134R/T159C and CoChR C108S and C108S/D136A—activated with 405-nm light and inactivated by coillumination with 594-nm light—and C1V1 E122T/C167S—activated by 561-nm light and inactivated by 405-nm light—were most promising in terms of highest photocurrents and efficient inactivation with coillumination. Although further engineering of step-function channelrhodopsin variants with higher photoconductances will be required to employ this approach at the nanoscale, our findings provide a framework to guide future development of this technique.


Introduction
Approaches to examine the biological mechanisms underlying synaptic plasticity rely on the artificial stimulation of neurons. Since its discovery, channelrhodopsin 2 (ChR2)-a channel that depolarizes neurons when activated by blue light-and its derivatives have become the dominant technique used to activate or inactivate neurons. 1 So far, the stimulation of neurons using channelrhodopsins has mainly focused on targeting specific cell populations or areas in the brain of rodents. 2,3 But the ability to stimulate subcellular nanodomains using channelrhodopsins 4 has not yet been extensively explored. Such a technique would be particularly advantageous to study aspects of synapse-specificity (where single synapses range in size from 0.04 μm 25 to ∼0.1 μm 3 for a dendritic spine) or the features of subsynaptic nanodomains.
Several attempts have been made to decrease the area of activation of channelrhodopsins. Spatially controlled stimulation of postsynaptic sites has been achieved by optogenetic stimulation of the presynaptic neuron, 6 for example. An activated area less than 30 μm in diameter was achieved by focused laser beams with reduced laser power, 7 and two-photon stimulation with 920-nm light has been used to evoke action potentials in individual cells. [8][9][10] However, so far, no technique exists that confines stimulation of light-gated channels to defined microscale-or nanoscale-subcellular regions. Focal activation of neurons could, in principle, be achieved using a stimulated emission depletion (STED)/reversible saturable optical fluorescence transitions (RESOLFT)-inspired illumination approach in which a focal light beam of one wavelength is used to activate channels, and a surrounding overlapping donut beam of another wavelength inactivates them. We tested such an approach by making use of mutations in light-gated channels that slow the photocycle, making it easier to control their switching between active and inactive states in response to two different wavelengths of light.
The photocycle of ChR2 has been intensively studied. [11][12][13][14] When ChR2 is illuminated with blue light, it undergoes a conformational change from its dark-adapted closed state (D470), through two nonconducting intermediate states (P500 and P390), and enters the open-channel state (P520). Subsequently, it passes through several intermediate states, until it finally reaches its dark-adapted closed state again and the cycle can be repeated. Interestingly, the P390 intermediate state and the P520 open state are sensitive to UV light (∼390 nm) and green light (∼520 nm), respectively, which mediate a direct transition to the closed-channel state (D470). 14 ChR2 channels that possess a mutation at residue C128-a conserved amino acid that interacts with the all-trans retinal Schiff base chromophorehave delayed inactivation and a prolonged open state following blue light activation, which can be converted directly to the closed state by green light, creating a so-called "step function" opsin. 15 The ChR2 C128S, C128T, and C128A step-function opsin mutants possess long open-channel states. These channels can be efficiently closed by exposure to a second closing wavelength (and opened again by an activating wavelength). However, the mutations that cause channels to close in response to a second wavelength of light often result in a reduction in photocurrent. 15 Focal activation of neurons by activating channelrhodopsins with one wavelength and inactivating them with an overlapping donut of another wavelength requires that channels have a high enough photocurrent to detect focal (micro-or nanoscale) activation and be efficiently inactivated by overlapping illumination of a distinct wavelength. Here, we tested channelrhodopsin variants for these properties.

Results
Focal stimulation of channelrhodopsins requires that activated channels remain relatively immobile in cell membranes. We therefore first tested the diffusion of channelrhodopsins expressed in dissociated hippocampal neurons. STED microscopy images of transfected dissociated hippocampal neurons revealed that ChR2-EYFP was mainly targeted to the plasma membrane of neurons [ Fig. 1(a)]. To examine channel diffusion, we performed fluorescence recovery after photobleaching (FRAP) experiments. A 2.5-μm region was illuminated with 488-nm light at 100% laser power for 1 s to bleach ChR2-EYFP in the illuminated region in neuronal processes or cell bodies and imaged for 200 s postbleaching [ Fig. 1(b)]. ChR2-EYFP fluorescence in the bleached region recovered partially, with a diffusion coefficient of 3.3 AE 0.2 × 10 −2 μm 2 ∕s in processes and 4.7 AE 0.02 × 10 −2 μm 2 ∕s in cell bodies, indicating low mobility, such that centrally activated channels would be inactivated by overlapping donut coillumination 500 nm in width or greater, assuming complete inactivation of step function opsin channels within 1 s. 15 A prerequisite for a potential micro/nanoscale focal illumination approach is that channels (1) have a relatively high photocurrent, to allow detection of focal activation, and (2) can be activated by one wavelength of light and inactivated by simultaneous overlapping illumination with another wavelength. Because high photocurrents and inactivation of channelrhodopsins with a second overlapping wavelength of light is difficult to predict based on structure-function analysis, we tested a broad range of channelrhodopsin variants for these properties. Dissociated hippocampal cultures were transfected with expression constructs of EYFP or mCherry-tagged light-gated ion-channels using calcium phosphate. Channel-specific lightevoked photocurrents were then tested to determine channel activation spectra, conductance, gating kinetics, and the ability to close channels with a second wavelength, using patch-clamp recordings of cells identified by their respective fluorescence. Spontaneous activity was suppressed by application of TTX, APV, CNQX, and gabazine, during recordings from neurons voltage-clamped at −70 mV. For each channel, we measured photocurrents elicited by 405, 488, 561, 594, and 639 nm single wavelength laser illumination. Subsequently, channel inactivation was tested by coillumination with all available laser combinations. Only the wavelength combinations for which effects were observed are shown in subsequent figures.
The first group of tested channels included the ChR2 variants with comparatively fast-photocycles: H134R, T159C, E123T/ T159C, and L132C. Fast photocycle channels do not possess a stable open-channel state and will open, close, and reopen in relatively fast succession. Upon initial exposure to their excitation wavelength, all channels open at once and as they proceed through their photocycles will finally reach an equilibrium (the stationary current phase) of constantly opening and closing channels, in which ∼60% of all illuminated channels are in the open-channel state, 16 inferred by the peak-to-stationary current ratios. The stationary current is very robust; the individual contribution of a single channel is small and of short persistence, because each individual channel closes quickly and reopens again. Therefore, illumination with a closing wavelength, exciting the conducting P520 or respective state, accelerating channel closure, is thought to have only a minor influence on the total photocurrent of fast photocycle channels. As expected, photocurrents of fast photocycle variants were generally strong and disappeared quickly when illumination ceased (Fig. 2). The most commonly used fast-photocyle channelrhodopsin variant contains the H134R mutation, which promotes enhanced stationary photocurrents. 17 This channel was activated by 405-and 488-nm light, yielding photocurrents of several hundred pA, and was not activated by wavelengths greater than 561 nm [ Fig. 2(a), top]. 594-nm light was previously reported to be the most efficient wavelength to close step function ChR2 channels. 15 H134R channels activated with 488-nm light showed a small but measurable reduction in photocurrent only in response to 594-nm coillumination. The photocurrent was reduced by ∼10% or ∼20% when channels were activated with 296 or 80 mW∕cm 2 488 nm light, respectively [ Fig. 2(a), middle]. Increasing the 594-nm laser power increased the inactivation accordingly [ Fig. 2(a), bottom].
T159C mutants are reported to possess increased photocurrents. 18 Photocurrents were induced by 405-and 488-nm light (and not further redshifted wavelengths) and were indeed approximately twice as large as those of H134R channels [ Fig. 2(b), top]. However, these channels showed barely perceptible inactivation with overlapping 405-or 594-nm illumination, when activated by either 488-nm light [ Fig. 2(b), middle] or 405-nm light [ Fig. 2(b), bottom]. Interestingly, the addition of the E123T mutation-which speeds up the photocycle and channel kinetics for temporal precision 18 -further increased peak and stationary currents to more than 1.5 nA and 700 pA, respectively, when channels were activated with 488-nm light and resulted in weak photocurrents of 250 pA when activated with 405-, 561-, and 594-nm light. This channel was inactivated slightly by 594-nm light following activation with 488-nm light [ Fig. 2(c), middle]. Due to its increased activation in the red spectrum, photocurrents of E123T/T159C elicited by 405-nm light increased even further with simultaneous illumination with 561-or 594-nm light [ Fig. 2(c), bottom].
The L132C ChR2 mutant also has high photocurrents and was reported to make channels calcium permeable, 19 which could be advantageous to assay focal activation by calcium imaging. This channel displayed strong photocurrents of 750 pA in response to 405-nm activation and 1.3 nA for 488-nm activation, with a reduced peak-stationary current ratio, compared to other channels. A pronounced slope in decay time indicates slow channel kinetics [ Fig. 2  The stabilized step-function opsin (SSFO) channel variant, ChR2 C128S/D156A, 11,15,22 has properties that make it attractive for a putative nanoscale focal activation approach; it is opened by blue-light and closed by green-light. This channel has been well characterized by spectral analysis of photocurrent responses to different wavelengths and has an optimal opening wavelength of 470 and optimal closing wavelength of 590. 15 We therefore used similar wavelengths to test coillumination. In single and coillumination tests, however, we found maximal photocurrents of only 40 to 60 pA with full field activation of transfected neurons, as expected [ Fig. 4(a), top]. In addition, 488-nm light-activated channels were only partially closed with coillumination of 561 [ Fig. 4(a), top] or 594 nm [ Fig. 4(a), bottom] light.
In an attempt to increase the photocurrent but maintain the switching capabilities of channels, we made a ChR2 variant with C128A/H134R/T159C mutations. We reasoned that H134R would enhance stationary photocurrents, 17 T159C would increase photocurrents, 18 and C128A would allow the channel to be switched to a closed state with 561-nm illumination (and would inactivate more quickly than the C128S mutation 22 ). This channel exhibited photocurrents of 350 to 400 pA in response to 405-or 488-nm illumination and weak photocurrents when illuminated at 561 nm. The photocurrent 23   was completely inactivated by single wavelength illumination with 594-nm light and partially inactivated by coillumination. This channel was inactivated even more efficiently (with 594nm light) when activated with 405-nm light [ Fig. 4(b)]. Thus, this channel appears more promising than the original SSFO channel for focal activation, in terms of larger photocurrent, and closing efficiency with inactivating light coillumination. We also generated a C128A/L132C/T159C channel, which should also have higher photocurrents and close with inactivating coillumination, and in addition be permeable to calcium, thus allowing calcium imaging to detect activation. This channel did indeed have similar high photocurrents and inactivation properties compared to the C128A/H134R/T159C channel, although its closing kinetics were slower [ Fig. 4(c)].
The C1V1 E122T/C167S channel is the corresponding step function version of the C1V1 channel. This channel maintained its redshifted wavelength activation properties and was activated by 488-, 561-, and 594-nm light, and weakly by 639-nm light. The C1V1 E122T/C167S channel showed efficient photocurrent reduction when stimulated with 561-nm light and coilluminated with 405-nm light, as indicated by earlier studies; 23 channels activated by 561-nm light could be efficiently closed by 405-nm light (but not by 488-nm light) [ Fig. 5(a)]. In contrast to the inactivating influence of 405-nm light on 561-nm activated currents, 405-nm coillumination following 488-nm light activation increased photocurrents further.
We further tested the addition of the switchable mutations (C108S and D136A, corresponding to C128S and D156A in ChR2) to the CoChR channel variants, which showed the largest photocurrents of all channels tested. (Chronos variants showed intracellular accumulation in neurons resulting in small photocurrents and were therefore not tested further.) The C108S mutation alone reduced the photocurrent of CoChR from ∼4 nA to 150 pA for 488-nm activation and to 50 pA for 405-nm activation but did indeed cause the channel to be partially inactivated by 594-nm light, following activation with 405-or 488-nm light [ Fig. 5(b)]. In addition, the CoChR C108S mutant exhibited 488-nm mediated channel closure, as seen from the photocurrent increase after stopping illumination. The addition of the D136A mutation to create CoChR C108S/D136A virtually eliminated this effect and increased 405-nm evoked photocurrents, which were inactivated to a greater extent by 594-nm light As opposed to other approaches, the eNPAC constructwhich expresses ChR2 H134R and NpHR3.0, a hyperpolarizing light-gated chloride pump, at equimolar amounts via connection by a P2A site that is cleaved intracellularly-does not depend on a specific illumination to close channels but rather on a balance between blue light activation of ChR2 H134R to depolarize cells and red light activation of the NpHR3.0 proton pump to hyperpolarize cells. 2 Photocurrent responses from eNPAC-expressing neurons were detected for all tested wavelengths (Fig. 6). 405and 488-nm evoked responses were almost identical to standard ChR2 H134R responses. However, 488-nm photocurrents displayed a small and brief transient after illumination ceased, most likely because NpHR3.0 is weakly activated by 488-nm light, and when this light is terminated the pumping of chloride-ions by NpHR3.0 stops more quickly than ChR2 H134R channels close, leading to a transient depolarization. This effect was not present for 405-nm excitation, implying that NpHR3.0 is not significantly activated by 405-nm light. When illuminated with 561-and 594-nm light, hyperpolarizing currents of ∼400 pA were recorded from eNPAC-expressing cells. Because ChR2 H134R is not activated by red wavelengths, these currents are predominantly from NpHR3.0 activation.
During initial channel tests of step-function opsins, we noted that even when 801 mW∕cm 2 (100%) 594-nm laser power was used, maximal channel inactivation was not reached. Therefore, we increased the 594-nm laser intensity by modifying the light path and exchanging the light guide from the DL594 laser module to the microscope with the beam splitter in the DirectFRAP unit (see Sec 4) and tested the effect of this modification on the most promising candidates inactivated by 594 nm light: ChR2 C128A/H134R/T159C and the CoChR C108S and C108S/D136 mutants (Fig. 7). ChR2 C128A/H134R/T159C channels were activated with 41.92 W∕cm 2 (10%) 488-nm laser power or 9.92 W∕cm 2 (20%) 405-nm laser power through the DirectFRAP laser path and inactivated by simultaneous illumination three consecutive times for 500 ms each, with 594-nm light of different intensities [ Fig. 7(a)]. The photocurrent values from these three exposures were pooled and compared to the average maximum photocurrent elicited by 500-ms single 488-or 405-nm illumination (between coillumination and a final 594-nm single wavelength exposure) to calculate % photocurrent reduction by coillumination with 594-nm light [ Fig. 7(b)]. In addition, activation intensities from 19.51 to 90.48 W∕cm 2 (1% to 20%) were tested against 129.75 W∕cm 2 (100%) DL594 power [ Fig. 7(c)]. Using the modified setup, we were able to reduce photocurrents of ChR2 C128A/H134R/ T159C elicited with 488-nm light by 54% and similar magnitude currents elicited with 405-nm light by 90%. Increased photocurrents were elicited by higher 488-nm activation intensities, which caused less efficient photocurrent reduction by coillumination. However, increased photocurrents elicited by higher [up to 9.92 W∕cm 2 (20%)] 405-nm activation were still efficiently reduced by coillumination.
We then tested 488-and 405-nm activation of CoChR C108S and CoChR C108S/D136A channels at varying intensities with maximal 594-nm inactivation [ Fig. 7(d)]. Photocurrent reduction was most efficient for 405-nm activation and 594-nm coillumination. For CoChR C108S, photocurrents were higher for 488-nm than for 405-nm light activation, and 488-nm light evoked photocurrents inactivated only slightly less efficiently than those elicited by 405-nm light. CoChR C108S/D136A, on the other hand, had slightly higher photocurrents elicited by 405-nm light than 488-nm light, which were eliminated with 594-nm coillumination, whereas 488-nm elicited photocurrents were only inactivated by ∼50%. A photocurrent increase when 488-nm illumination is discontinued was visible for the CoChR channel and especially for the C108S mutant. In general, currents of the CoChR step function variants were weaker than the ChR2 step function variants and did not increase when higher 405-nm light intensities were used. However, 405-nm evoked currents could be efficiently reduced by exposure to 594-nm light. These experiments suggest that 405-nm light is the ideal activation wavelength for experiments where strong photocurrents and efficient photocurrent reduction, using 594nm coillumination, is needed.
A summary of all channels tested in terms of activating/inactivating wavelengths, magnitude of photocurrent, and degree of inactivation is shown in Fig. 8. The fast photocycle variants H134R, T159C, E123T/T159C, L132C, C1V1 E122T/E162T, ReaChr, CoChR, and Chronos all showed only ∼1% to 20% inactivation by coillumination [ Fig. 8(a)  laser intensity increased the extent of inactivation, but decreased overall photocurrent. Raising inactivation laser intensity also increased the extent of inactivation, only slightly for these channel variants. The CoChR channel had two to six times higher photocurrent than other channels, with ∼15% to 20% inactivation. The original stable step-function opsin variant C128S/ D156A exhibited 33% inactivation with coillumination but a photocurrent of only ∼50 pA. The addition of the C128A switchable mutation to channels with H134R/T159C or L132C/T159C mutations reduced photocurrents slightly but increased the extent of inactivation, especially by 405-nm light. The addition of the C167S switchable mutation to the C1V1 channel did not reduce the photocurrent (elicited by 561-nm light) but did increase the extent of inactivation (by 405-nm light) to ∼84%. Adding the C108S or C108S/D136A switchable mutations to CoChR significantly reduced the photocurrent, from ∼2000 to 200 pA, but improved channel inactivation. eNPAC ChR2 photocurrents were completely inactivated by coillumination to activate NpHR3.0. In further tests of the most promising candidates, e.g., C128A/H134R/T159C and CoChR C108S or C108S/D136A channels [ Fig. 8(b)], we found that both the photocurrent and efficiency of inactivation plateaued. 405-nm light activation increased inactivation efficiency compared with 488-nm light. In general, photocurrent magnitude and degree of inactivation were inversely related, and both photocurrent strength and inactivation efficiency plateaued and were not further increased by increasing activation/inactivation light intensity.
To estimate the size of illuminated regions for which photocurrents can be detected, we tested focal single wavelength 488-nm light activation of proximal neuronal processes of cells expressing the ChR2 T159C channel. Photocurrents elicited by illumination of membrane areas with diameters down to 1.2 μm could reliably be detected using electrophysiological patch-clamp recordings in the whole-cell configuration [ Fig. 9(a)]. However, detectable photocurrents were already weak, suggesting that photocurrents from smaller areas will be beyond detection limits, especially for switchable channel variants, which have reduced photocurrents. We therefore first investigated the feasibility of nanoscale optogenetic stimulation using a microscale approach and an illumination strategy borrowed from super-resolution microscopy, in which a central area is illuminated with a wavelength of light that activates channelrhodopsins while an overlapping donut region is simultaneously illuminated with a wavelength of light that inactivates these channels [ Fig. 9(b)], thereby confining activation to a small central region. To accomplish this, we used photoactivation masks for 405-or 488-nm illumination of 16.7-μm diameter areas, in combination with a donut-shaped 594-nm inactivation mask, resulting in a central activated region of 6.2 μm in diameter. We first verified illumination regions using a fluorescently labeled slide [ Fig. 9(c)]. We then tested the approach on neurons transfected with eNPAC, a construct that coexpresses equimolar amounts of ChR2 H134R and NpHR3.0 2 . In neurons transfected with eNPAC, coillumination with 594-nm light effectively inactivated photocurrents evoked by 488-nm light [ Fig. 9(d) In a second approach, the same illumination principle was applied to the ChR2 slow-photocyle variant C128A/H134R/ T159C [ Fig. 9(e)]. The respective evoked photocurrents for 6.2 and 16.7 μm wide areas, illuminated with 405-nm light, were 50 and 150 pA. When 16.7 μm 405-nm photostimulation was combined with an overlapping donut of 594-nm illumination, photocurrents could be reduced to values close to those evoked by stimulation of a 6.2-μm area alone, independent of the order in which the different wavelengths were applied [ Fig. 9(e)].

Discussion
We investigated an approach to focally confine channelrhodopsin activation using switchable light-gated channels and an illumination approach inspired by STED/RESOLFT microscopy. Of all tested candidates, the ChR2 C128A/H134R/T159C and the slow CoChR variants C108S and C108S/D136A-activated with 405-nm light and inactivated by coillumination with 594nm light-and the C1V1 E122T/C167S channel-activated by 561-nm light and inactivated by 405-nm light-were most promising in terms of highest photocurrents and efficient inactivation with coillumination. The combination of coexpression of a depolarizing cation-channel and hyperpolarizing light-gated chloride-pump (eNPAC) also represents an interesting alternative, in which counterbalancing currents can be used to cause a net voltage change of 0 mV in the area of 405-or 488-, and 594-nm coillumination. Based on our experiments, three approaches using lightgated ion-channels/pumps could be used to achieve focal photostimulation. The first is simultaneous activating wavelength illumination of a central region and inactivating wavelength coillumination in a donut overlapping this central activated region. The second (which could be used with step-function opsin channels that do not show sufficient inactivation during coillumination) is fast sequential illumination, in which channels are opened with one wavelength and subsequently closed in an outer donut region. For this approach, the time between initial channel opening and closing of the outer channels should be as short as possible. The third option makes use of the eNPAC construct to control depolarizing and hyperpolarizing currents with different wavelengths of light in central and   surrounding regions. Theoretically, the wavelengths can also be inverted, producing highly focal areas of hyperpolarization, mimicking inhibitory synapses. However, the use of eNPAC is not ideal for nanoscale optogenetics, since (1) coactivation of ChR2 and NpHR3.0 will reduce membrane resistance and may shunt physiological inputs, (2) simultaneous Na þ and Cl − import may lead to osmotic changes in the cell (although these effects should be minimal with focal activation), (3) the efficiency of activation/inactivation may be variable given that a 1:1 stoichiometric expression of ChR2 and NpHR3.0 cannot be guaranteed, due to differences in rates of folding, trafficking, and degradation, and (4) activation/inactivation will depend on membrane voltage since ChR2 is an ion channel with reversal potential near 0 mV, and NpHR is a pump whose current depends only weakly on voltage. Efficient inactivation of channels with coillumination usually required reducing activation laser intensities. It was reported that 594-nm inactivating light should be roughly 1000-fold more intense than 488-nm activating light to achieve photocurrentinactivation of ∼95% for ChR2 C128S. 24 In our experiments, we tested the lowest available laser power for activation (1%; 4.48 W∕cm 2 ). The power density could be further reduced using filters or beam splitters to achieve 1000-fold higher 594-nm power densities; however, this would mainly reduce photocurrents from activated channels and not lead to a more efficient channel gating. Interestingly, channels were more efficiently inactivated by 594-nm light following 405-nm light activation, than following 488-nm light activation, even at only ∼100-fold higher 594-nm light power densities. We assume that 405-nm light illumination activates the P470 open state but does so less efficiently. However, it is also possible that these channels have a separate conducting state for stimulation in the UV spectrum, which is more easily reduced by 594-nm illumination. In any case, 405-nm activation was superior to 488-nm activation, for subsequent inactivation by coilluminating 594-nm light to close channels. For eNPAC, 405-nm light evoked ChR2-mediated depolarization was completely counterbalanced by 80 to 160 mW∕cm 2 594-nm illumination to activate hyperpolarizing NpHR3.0. Although these values fluctuated between experiments, they were minimal, given approximate equimolar expression of ChR2 and NpHR3.0 in eNPAC. In this case, 405-nm activation light (rather than 488 nm) also avoids currents that arise from NpHR3.0 bluelight activation. An important question is whether current optogenetic tools are powerful enough to allow detection at the nanoscale. Photocurrent recordings from Xenopus oocytes have yielded estimated single channel conductances of ∼50 fs for wildtype ChR2, with single channel currents of ∼5 fA. 25 Using nonstationary noise analysis, single channel Na þ conductances of wild-type ChR2 and ChR2 H134R in HEK cells were determined to be 41.5 fs at 200 mM extracellular Na þ , with single channel currents of 3.5 fA at −60 mV. 16 Other studies using nonstationary noise analysis estimate ChR2 single channel conductance at around 1 ps, which principally would allow single channel recordings. 26 However, the absence of such published recordings to date suggest this value may be an overestimation. During the stationary current photocycle phase, ∼60% of ChR2 H134R channels are expected to be in the open-channel state. 16,17 Thus, ∼226 channels are active per μm 2 cell membrane in HEK cells. 16 If the proposed channel density in HEK cells translates to a similar presence in neuronal membranes, stimulation of a synapse size area (0.04 μm 2 ) would lead to the opening of approximately nine channels and currents   When a 16.7-μm 488-nm illuminated region was combined with a 594-nm overlapping donut region (leaving a central 6.2 μm region of activation), the photocurrent is reduced to a level similar to that evoked by a 6.2-μm region alone.
Neurophotonics 015007-11 Jan-Mar 2019 • Vol. 6(1) of 31.5 to 45 fA at −60 mV-too low to be detected using standard electrophysiological methods, or currently available calcium or voltage sensors that can be imaged at wavelengths that do not influence light-gated channels. 27,28 An increase in channel abundance in the membrane, however, e.g., by higher expression levels or targeting motifs to synapses, or an increase in single channel conductance could enable detection of activation at the nanoscale. To mimic synaptic activation, ideally channels would possess properties that match currents elicited by AMPA receptors at excitatory synapses, where 1 to 2 AMPA receptors open during the peak of the postsynaptic current. 29,30 Single channel AMPA conductance is ∼8 to 10 ps. 30,31 To match currents of a single AMPA receptor, the conductance of a wildtype ChR2 would need to be enhanced roughly 200-fold. Lightgated genetically modified ionotropic glutamate channels (LiGluRs), which are based on AMPA receptors and can be activated and inactivated by two different wavelengths of light, are another alternative. 32,33 LiGluR steady-state currents were reported to be fivefold larger than those of wild-type ChR2, 34 i.e., on the order of Chronos currents, but LiGluR requires the addition of an exogenous chemical photoswitch and may also alter the native firing properties of cells in which it is expressed since it is based on the glutamate receptor that is natively present in the brain.
Although we demonstrate the feasibility of a focal optogenetics approach at the microscale, we also show that at present channel conductance is too low to allow nanoscale activation. Nevertheless, this work may be useful for future development of nanoscale optogenetics. It is likely that stronger conducting channel variants will be found. New single channel optogenetic tools with faster switching kinetics that allow depolarization and hyperpolarization could allow nanoscale activation. For instance, the ion selectivity of ChR2 has recently been selectively modified to create a chloride-conduction channel. 35,36 It would also be interesting to test the sdChR C138S/E154A variant, because it has been reported to possess good photocurrent inhibition. 24 In addition, optogenetics now extends beyond light-gated channels. Inducing protein conformational changes or interactions in nanodomains by light, for example, may be feasible using this technique.

Animals
Use of animals for experimentation was approved and performed according to the specifications of the Institutional Animal Care and Ethics Committees of Goettingen University (T10.31) and of the German animal welfare laws.

Dissociated Primary Hippocampal Cultures
Dissociated hippocampal cultures were cultured on 25-mm coverslips (Paul Marienfeld GmbH; cat. no. 0117650) coated with 0.04% polyethylenimine (PEI) (Sigma; cat. no. P3143) in sixwell plates (CytoOne; cat. no. CC7672-7506); 2 ml of 0.04% PEI in dH 2 O, stored at −20°C was added per well at room temperature for ∼14 h, followed by three washes with dH 2 O after which plates were directly used for cultures or stored in dH 2 O at 4°C until needed.

Calcium Phosphate Transfection
On DIV3, conditioned culture medium was collected and exchanged for pre-equilibrated (37°C and 5% CO 2 ) Opti-MEM (Gibco; cat. no. 11058-021). One-third fresh culture medium was added to the collected conditioned medium that was filtered through a 0.22-μm syringe-driven filter (Millipore; cat. No. SLGV033RS), and stored in the incubator. For each well of a six-well plate, 7.5-μl 2 M CaCl 2 and 3.5 to 4 μg plasmid DNA in 60-μl dH 2 O were mixed and 60-μl transfection buffer (274 mM NaCl, 10 mM KCl, 1.4 mM Na 2 HPO 4 , 15 mM glucose, 42 mM Hepes, pH 7.05 to 7.12) was then added dropwise, under gentle agitation. This solution was incubated for 20 min at room temperature and then gently added to cells, which were returned to the incubator for 90 min. The Opti-MEM plus transfection mix was then exchanged for 37°C and 10% CO 2 pre-equilibrated neurobasal medium, returned to the incubator for 10 min, and subsequently exchanged for the stored conditioned medium. before and after each experiment by a brief illumination with the respective closing wavelength. Channelrhodopsins were characterized by their responses to 500-ms light pulses of 405, 488, 561, 594, and 639 nm, using the DL594 laser and no photomask for the 594 nm and the TIRF laser for the other wavelengths. To test focal optogenetic stimulation and record responses, 488-nm light pulses were delivered using DirectFRAP photomasks and the 100× objective; 594-nm Rapp Opto customdesigned photomasks were overlaid to produce a coillumination donut surrounding a central region.
Microscope and components including lasers were controlled with Zeiss AxioVision and Zen Blue software. Transfected cells were identified by fluorescence and selected based on health and membrane integrity. Recordings were acquired from cells voltage-clamped at −70 mV. Liquid junction potential, pipette, and cell capacitance influences were compensated. Recordings were managed with IGOR Pro (Wavemetrics; version 6.22A) and the Patcher's Power Tools extension for HEKA files provided by the Department of Membrane Biophysics at the Max Planck Institute for Biophysical Chemistry in Goettingen. In cases where small shifts in timing of laser pulses occurred, traces were manually aligned to illumination onset for comparison.

Disclosures
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.