Cellular resolution circuit mapping with temporal-focused excitation of soma-targeted channelrhodopsin

Christopher A Baker; Yishai M Elyada; Andres Parra; M McLean Bolton

doi:10.7554/eLife.14193

. 2016 Aug 15;5:e14193. doi: 10.7554/eLife.14193

Cellular resolution circuit mapping with temporal-focused excitation of soma-targeted channelrhodopsin

Christopher A Baker ^1,^*, Yishai M Elyada ^2,^†, Andres Parra ², M McLean Bolton ^1,^*

Editor: Michael Häusser³

PMCID: PMC5001837 PMID: 27525487

Abstract

We describe refinements in optogenetic methods for circuit mapping that enable measurements of functional synaptic connectivity with single-neuron resolution. By expanding a two-photon beam in the imaging plane using the temporal focusing method and restricting channelrhodopsin to the soma and proximal dendrites, we are able to reliably evoke action potentials in individual neurons, verify spike generation with GCaMP6s, and determine the presence or absence of synaptic connections with patch-clamp electrophysiological recording.

DOI: http://dx.doi.org/10.7554/eLife.14193.001

Research Organism: Mouse

eLife digest

Nerve cells called neurons carry information around the body in the form of electrical impulses and pass signals to each another to form circuits that link different organs and tissues. Mapping out the neurons in the brain can reveal how different circuits contribute to an animal’s behavior. Yet, because the brains of mammals contain millions of neurons, these circuits are often difficult to untangle.

One way to tease apart circuits of neurons uses a technique called optogenetics, which involves manipulating the genes inside neurons such that the cells produce a light-sensitive protein and respond to blasts of light. The aim is to activate a specific neuron and then see which other neurons are activated shortly afterwards, revealing a connected circuit. However, exposure to light can be imprecise. Also, the neurons in the brain are so densely packed that the nerve endings from neighboring neurons often overlap without actually being connected. This makes it unclear if activated neurons are truly part of the same circuit or simply bystanders reacting to the same nearby blast of light.

To overcome this limitation, Baker et al. developed a new optogenetic approach with two important features. First, the approach makes use of a light-sensitive protein called channelrhodopsin that had been modified to confine it to the cell body of each neuron and exclude it from the nerve endings. Second, pulses of laser light were specifically shaped to target only the cell body of an individual neuron. Baker et al. show that this new method can activate neurons inside slices of mouse brain without affecting the neighboring neurons. This allowed circuits of neurons to be mapped in fine detail.

This new optogenetic method is expected to shed light on the patterns of nerve signals that contribute to animal behavior. The approach may also be modified to use other light-sensitive proteins or investigate how neural circuits are altered in animal models of human disorders like autism and schizophrenia.

DOI: http://dx.doi.org/10.7554/eLife.14193.002

Introduction

The synaptic organization of individual neurons into circuits is the physiological basis for the interpretation of sensory input and production of behavioral responses. Understanding the precise patterns of connectivity among the distinct types of neurons that comprise neural circuits is critical for elucidating circuit function and ultimately requires methods that can map functional connectivity with single-cell resolution. Optical activation of single neurons using two-photon excitation of caged neurotransmitters or optogenetic probes such as channelrhodopsin (ChR2) provides a powerful approach for assessing the synaptic connections of single neurons. In particular, optogenetic mapping utilizing ChR2 and the rapidly expanding family of opsin variants have increased the flexibility and precision of mapping paradigms. Variations in the single-channel properties of the opsins can be exploited to generate rapid action potential trains or sustained depolarizations (Mattis et al., 2012), and new red-shifted variants have facilitated excitation deeper in tissue and have enabled simultaneous optical control of two distinct neuronal populations (Klapoetke et al., 2014; Lin et al., 2013; Yizhar et al., 2011). In addition, genetic restriction of opsin expression using transgenic mouse lines enhances the ability to activate and assess the connectivity of specific cell types.

Despite the great potential of optogenetics for mapping the synaptic connections of single neurons, there are multiple issues that have limited its effectiveness. First, two-photon activation of single neurons with ChR2 is complicated by its kinetics and low single-channel conductance. A diffraction-limited spot does not activate sufficient channels simultaneously to reliably bring neurons expressing ChR2 to action potential threshold. Several solutions have been implemented to address this. Rapid scanning of a diffraction-limited two-photon excitation spot across an opsin-expressing cell allows sufficient temporal integration to generate action potentials (Packer et al., 2012; Prakash et al., 2012; Rickgauer and Tank, 2009). Alternatively, scanless two-photon excitation by temporal focusing (Oron et al., 2005; Zhu et al., 2005) increases the number of simultaneously excited opsin molecules by expanding the beam in the imaging plane without sacrificing the optical sectioning of multiphoton microscopy (Andrasfalvy et al., 2010; Losonczy et al., 2010; Papagiakoumou et al., 2010; Rickgauer et al., 2014). The implementation of diffractive optical elements (Fino and Yuste, 2011; Nikolenko et al., 2007; Packer and Yuste, 2011) or spatial light modulators (Dal Maschio et al., 2010; Nikolenko et al., 2008; Packer et al., 2012, 2015; Papagiakoumou et al., 2010, 2009, 2008) has also allowed for more complicated excitation profiles that encompass multiple spots around a cell, ensembles of neurons, or particular branches of dendritic trees.

While these approaches have made it possible to provide sufficient two-photon illumination to reliably drive action potentials, the ability to use optogenetic stimulation to selectively target single neurons remains challenging because the opsins are expressed throughout the dendritic and axon terminal fields, generating a potential confounding source of light-induced electrical responses. For example, it can be difficult to know whether a recorded electrophysiological event is due to stimulation of a presynaptic cell or 'direct' stimulation of a portion of the recorded cell’s dendritic arbor. Although these possibilities can be distinguished by their kinetics, large amplitude direct responses may obscure simultaneous smaller synaptic events. This essentially leaves an indeterminate region of any circuit 'map' coinciding with the dendritic arbors of the recorded neuron, which can extend for 200 µm or more around the soma. In addition, the optogenetic approach could be compromised by unintended activation of fibers of passage or local axonal boutons, which are known to respond to temporal focusing of two-photon excitation (Andrasfalvy et al., 2010; Losonczy et al., 2010). Exclusion of opsins from axonal compartments has previously been achieved by fusing the opsin with targeting domains that bind to myosin Va motors necessary for transporting proteins into dendrites; one such motif from melanophilin is sufficient to exclude ChR2 from axons and enhance the resolution of neural circuit maps (Lewis et al., 2009). The resulting ChR2 distribution, however, remains throughout the dendritic tree and thus does not solve the problem of undesired direct activation of a neuron’s processes while trying to stimulate other neurons in close proximity.

To overcome these limitations, we combined temporal focusing with spatial confinement of ChR2 expression to the neuronal cell body and proximal dendrites. Our alternative targeting approach took advantage of the Kv2.1 potassium channel, which has a particularly unique localization to clusters at the neuronal soma and proximal dendrites (Trimmer, 1991) achieved through a 66-amino acid domain in its C-terminus (Lim et al., 2000) that drives its association with myosin IIb and specific post-Golgi transport vesicles destined for the somatic compartment (Jensen et al., 2014). Furthermore, this targeting signal is sufficient to alter ChR2 trafficking in retinal ganglion cells (Wu et al., 2013). We have employed this approach to target ChR2 to the soma and proximal dendrites of neurons in somatosensory cortex and added a nuclear fluorescent tag to identify ChR2-expressing neurons for targeting a two-photon temporal focused mapping beam. We combined this soma-targeted ChR2 with verification of successful action potential generation with a genetically encoded calcium indicator in our mapping protocol. Our approach allows robust and precise activation of neurons in brain slices for the construction of functional synaptic connectivity maps with single-cell resolution without loss of information about local connections in the region of the dendritic arbor of the recorded neuron or inadvertent activation of axons.

Results

We stimulated neurons expressing soma-targeted ChR2 in acute slices of mouse cortex using scanless temporal focusing (TF), which has been used successfully for optogenetic stimulation at high axial resolution in scattering tissue samples (Andrasfalvy et al., 2010; Losonczy et al., 2010; Papagiakoumou et al., 2010, 2013; Rickgauer et al., 2014). TF uses a diffraction grating (Figure 1—figure supplement 1) to separate the spectral components of a pulsed laser beam, resulting in a temporally broadened pulse that is inefficient at excitation except at the focal plane, where the components are recombined. This yields a volume of excitation in which the diameter in the x-y plane and the thickness in the axial plane are controlled independently (Oron et al., 2012, 2005). We designed our excitation volume to approach the dimensions of a typical neuronal soma (Figure 1—figure supplement 2).

To restrict expression of ChR2 to the soma and proximal dendrites, we generated ChR2 fusion proteins by attaching a 65 amino acid motif from the Kv2.1 voltage-gated potassium channel to the carboxy terminus of ChR2-EYFP (Lim et al., 2000). Nontargeted ChR2-EYFP fluorescence was distributed throughout the processes of dissociated cortical neurons that had been filled with a fluorescent dye (Figure 1A). In contrast, targeted ChR2-EYFP-Kv2.1 was located primarily in the soma and excluded from the axon and distal dendrites (Figure 1A).

Figure 1. — (A) Fluorescence of ChR2-EYFP (green) in live dissociated cortical neurons that had been patched and filled with Alexa 594 dye (magenta) as visualized by two-photon microscopy. Scale bar = 100 µm. (B) Representative two-photon maximum intensity projections of Alexa 594 fluorescence and current responses to a single 150 ms TF stimulation pulse (red bar) for patched and dye-filled pyramidal cells in acute slices expressing nontargeted (N) or targeted (T) ChR2. Scale bar = 100 µm. (C) Mean (solid lines) ± s.e.m. (shaded regions) for current responses to TF stimulation measured at specific points along a single dendrite (N=10 nontargeted cells and 13 targeted cells). Fitting each curve with an exponential function demonstrated that the nontargeted and targeted datasets were significantly different (*p<0.05 by F-test). (D) Maps of direct currents in representative cells expressing ChR2 constructs when stimulating a 15 × 15 grid of locations. Each pixel in a map represents the direct current observed in the patched cell when that location in the slice was stimulated. Each position in a map was stimulated with the minimum power that evoked action potentials in 10/10 trials when stimulation was applied to the soma. Currents evoked by such powers ranged from 20 to over 250 pA, as indicated by the scale at bottom. Scale bar at lower left = 100 µm.

**DOI:** http://dx.doi.org/10.7554/eLife.14193.003

Figure 1—figure supplement 1. — (A) Fluorescence of ChR2-EYFP (green) in live dissociated cortical neurons that had been patched and filled with Alexa 594 dye (magenta) as visualized by two-photon microscopy. Scale bar = 100 µm. (B) Representative two-photon maximum intensity projections of Alexa 594 fluorescence and current responses to a single 150 ms TF stimulation pulse (red bar) for patched and dye-filled pyramidal cells in acute slices expressing nontargeted (N) or targeted (T) ChR2. Scale bar = 100 µm. (C) Mean (solid lines) ± s.e.m. (shaded regions) for current responses to TF stimulation measured at specific points along a single dendrite (N=10 nontargeted cells and 13 targeted cells). Fitting each curve with an exponential function demonstrated that the nontargeted and targeted datasets were significantly different (*p<0.05 by F-test). (D) Maps of direct currents in representative cells expressing ChR2 constructs when stimulating a 15 × 15 grid of locations. Each pixel in a map represents the direct current observed in the patched cell when that location in the slice was stimulated. Each position in a map was stimulated with the minimum power that evoked action potentials in 10/10 trials when stimulation was applied to the soma. Currents evoked by such powers ranged from 20 to over 250 pA, as indicated by the scale at bottom. Scale bar at lower left = 100 µm.

**DOI:** http://dx.doi.org/10.7554/eLife.14193.003

As a functional assay to compare the distribution of ChR2 in the soma and dendrites of targeted and non-targeted constructs, we used TF stimulation at intervals along the apical dendrite of patched layer II/III pyramidal neurons in acute coronal brain slices of somatosensory cortex from mice expressing opsins from viral constructs and recorded light activated currents in voltage clamp (Figure 1B). The stimulation power for these experiments was determined independently for each neuron to be the minimum that elicited a single action potential in 10 out of 10 trials when the TF spot was placed directly over the soma; this value was 0.92 ± 0.24 mW/µm² for targeted ChR2 and 1.65 ± 0.26 mW/µm² for the nontargeted version, owing to the increased sensitivity of the targeted construct (see below). Dendrites were followed throughout the depth of the tissue and areas were selected for stimulation such that the dendrite was planar throughout the extent of the 10 µm TF disc. The TF-stimulated current declined ~10-fold at 50 µm from the soma with the targeted construct, versus two fold for the non-targeted construct (Figure 1C; N=10 untargeted cells from four animals and N=13 targeted cells from six animals). The reduced dendritic ChR2 in the targeted construct opens up the possibility of using light stimulation with patch recording to identify synaptic currents that originate from nearby neurons. To illustrate this point, we compared the spatial distribution of direct currents recorded in Layer II/III pyramidal neurons in acute slices of somatosensory cortex expressing non-targeted and targeted constructs while sequentially stimulating points at 20 µm intervals over a 300 × 300 um grid surrounding the soma. Stimulation of cells expressing non-targeted ChR2-evoked currents up to 200 µm away from the neuronal soma, often delineating a pattern indicative of the dendritic arbor of the cell (Figure 1D). In contrast, cells expressing the targeted construct exhibited significant currents only when stimulated within the 25–50 µm immediately adjacent to the cell body. The lack of direct light-activated currents throughout much of the neuron’s dendritic field makes it possible to visualize synaptic currents that would be evoked by ChR2 activation of nearby presynaptic neurons, even those that lie within the neuron’s dendritic field.

In brain sections from mice injected with viruses encoding ChR2 constructs, examination of regions near the edge of the extent of virus-driven expression revealed markedly denser labeling of neuronal processes around a single-neuron expressing nontargeted ChR2 versus the area around several neurons expressing the targeted version (Figure 1—figure supplement 3A). We also saw processes reminiscent of axons at distances of several hundred microns away from the cell bodies of conventional ChR2-positive neurons. To functionally characterize the extent of axonal opsin expression, we again patched layer II/III pyramidal neurons in acute coronal brain slices of somatosensory cortex and recorded light-activated currents in voltage clamp while stimulating multiple positions along putative axonal processes, which were distinguished by their thin profile, absence of spines, and presence of occasional varicosities. Although the amount of detected current decreased rapidly for both constructs as TF stimulation was moved along the axon, noticeable currents could be detected at distances of 50 µm or greater from the soma of cells expressing nontargeted ChR2 (Figure 1—figure supplement 3B). In contrast, cells expressing targeted ChR2 demonstrated an 80% reduction of current when stimulating just 20 µm down the axon and significantly less current than cells expressing nontargeted ChR2 throughout its examined length (Figure 1—figure supplement 3C).

Interestingly, the targeted ChR2 also exhibited increased peak current amplitude in response to TF activation, presumably by concentrating channel density at the soma (Figure 2A; from 308.3 ± 43.5 pA in 12 nontargeted cells to 760.5 ± 146.0 pA in 12 targeted cells at 3.63 mW/µm²; p=0.0101 for main effect of targeting by two-way repeated measures ANOVA), leading to an over three-fold reduction of the power required to reach action potential threshold (Figure 2B; from 2.75 ± 0.31 mW/µm² in 17 nontargeted cells to 0.88 ± 0.23 mW/µm² in 19 targeted cells; p=8.028 × 10^–5 by Mann-Whitney U Test). At the threshold stimulation power for each cell, the amount of evoked current was similar between targeted and untargeted constructs (261.06 ± 31.62 pA for untargeted ChR2 and 222.70 ± 27.21 pA for targeted ChR2 p=0.3677 by two sample t-test), consistent with the observation that the targeting modification had no effect on rheobase, other intrinsic physiological parameters, or action potential properties in response to current injections (Figure 2—figure supplement 1 and Table 1).

Figure 2. — (A) Evoked currents in acute slices in response to 150 ms TF stimulation of increasing intensity; targeted ChR2 exhibited significantly greater current (two-way repeated measures ANOVA, p=0.0101 for main effect of targeting, p=0.0056 for targeting × power interaction, *p<0.05 Holm-Bonferroni post hoc test). (B) Minimum TF power required to elicit a single action potential. Each point indicates a single cell; cells expressing targeted ChR2 had a much lower threshold (*p=8.028 × 10^–5, Mann-Whitney U Test). (**C–D**) Probability of evoking spikes per 10 trials in cells expressing targeted (red) or nontargeted (blue) ChR2 when stimulating the soma and at laterally and axially displaced locations.

**DOI:** http://dx.doi.org/10.7554/eLife.14193.007

Figure 2—figure supplement 1. — (A) Evoked currents in acute slices in response to 150 ms TF stimulation of increasing intensity; targeted ChR2 exhibited significantly greater current (two-way repeated measures ANOVA, p=0.0101 for main effect of targeting, p=0.0056 for targeting × power interaction, *p<0.05 Holm-Bonferroni post hoc test). (B) Minimum TF power required to elicit a single action potential. Each point indicates a single cell; cells expressing targeted ChR2 had a much lower threshold (*p=8.028 × 10^–5, Mann-Whitney U Test). (**C–D**) Probability of evoking spikes per 10 trials in cells expressing targeted (red) or nontargeted (blue) ChR2 when stimulating the soma and at laterally and axially displaced locations.

**DOI:** http://dx.doi.org/10.7554/eLife.14193.007

Table 1.

Intrinsic electrophysiological properties of neurons expressing normal or targeted channel rhodopsin constructs. Rheobase measurements were made in response to current injections. All measurements are mean ± s.e.m. No significant differences as an effect of targeting were found (Mann-Whitney U Tests, all p>0.05).

DOI: http://dx.doi.org/10.7554/eLife.14193.011

	Nontargeted	Targeted	p
Input resistance (MΩ)	129.16 ± 8.91 (N = 11)	148.36 ± 17.76 (N = 18)	0.9462
Capacitance (pF)	125.25 ± 16.99 (N = 11)	89.85 ± 7.66 (N = 18)	0.0887
Resting potential (mV)	−72.71 ± 3.86 (N = 7)	−76.87 ± 1.87 (N = 8)	0.3532
Rheobase (pA)	160.00 ± 7.69 (N = 12)	147.39 ± 12.92 (N = 23)	0.5357
Spikes at 1.5× rheobase	7.18 ± 0.52 (N = 12)	7.56 ± 0.83 (N = 23)	0.8377

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The ability to trigger action potentials at lower incident power, coupled with the somatic restriction of the targeted construct, should provide enhanced spatial and temporal resolution for mapping neuronal circuits. We measured the spatial resolution of action potential generation by moving the TF spot to different lateral and axial locations relative to a patched neuron expressing targeted ChR2 and examining the proportion of 10 trials that resulted in an action potential when using the threshold stimulation intensity. The full-width at half maximum of these measurements was 11.1 µm laterally and 23.3 µm axially (N=10 cells from 7 animals; Figure 2C and D). Similar measurements using nontargeted ChR2 demonstrated resolution of 19.6 µm laterally and 36.2 µm axially (N=13 cells from 5 animals; Figure 2C and D); both curve fits were significantly different from the targeted ChR2 versions (p<0.05 by F-test). In terms of temporal precision, the mean latency from light onset to generation of a single action potential in the above experiments was 38.98 ± 17.33 ms; the average jitter (defined as the standard deviation of the latency across the ten trials for a given cell) was 6.8 ± 2.1 ms. Increasing stimulation power beyond threshold provoked additional action potentials and eventually shortened the latency to first spike to 9.3 ± 1.8 ms (Figure 2—figure supplement 2), consistent with reported values for stimulating cortical neurons with sculpted light (Papagiakoumou et al., 2010). Targeted ChR2-expressing cells exhibited shorter action potential latencies than nontargeted ChR2 cells at equivalent stimulation intensity, consistent with latency being power-dependent and the earlier observation that targeted ChR2 is more sensitive. Although we concentrated efforts on longer stimulation pulses to increase the number of spikes and facilitate calcium imaging (see below), we verified the performance of targeted ChR2 under a shorter stimulation regimen. Temporal focusing pulses of 32 ms were effective at generating action potentials in opsin-expressing cells, and targeted ChR2 exhibited greatly enhanced photocurrents and lower power thresholds under these conditions (Figure 2—figure supplement 3). Moreover, the ability to reduce latency to less than 10 ms with increased power (Figure 2—figure supplement 2) suggests that even shorter stimulation pulses may still be effective. Therefore, the combination of temporal focusing and soma-targeted ChR2 expression yields a highly reliable and spatially precise means to stimulate action potentials with somatic illumination.

To demonstrate the general utility of these techniques for mapping synaptic connections, we performed connectivity experiments in acute somatosensory cortical slices. We generated a bicistronic adeno-associated virus (AAV) construct encoding targeted ChR2-Kv2.1 followed by a P2A ribosomal skipping sequence and a histone 2B-mRuby2 fusion protein to fluorescently label neuronal nuclei to identify cells for TF stimulation. We patched a cell in layer II/III, and then stimulated surrounding cells that had been identified by nuclear mRuby2 fluorescence. Action potential firing in a light-stimulated neuron was verified by an increase in the fluorescence signal measured with the genetically encoded calcium sensor GCaMP6s (Chen et al., 2013) expressed from a separate AAV construct. For these experiments, we used an average stimulation power (2.29 ± 0.55 mW/µm²) that, when combined with a 150 ms stimulation pulse, frequently led to trains of 2–4 action potentials in the patched cells and facilitated detection of larger calcium transients. We used an imaging power and dwell time that did not lead to action potential generation in any cells recorded, instead causing an average inward current of 29.5 ± 9.5 pA (N = 7 cells from 5 animals)—well below the average rheobase. The ability to identify a ChR2-Kv2.1-expressing neuron for TF stimulation by the presence of a fluorescent nuclear label and verify that the neuron has in fact been stimulated successfully by detecting calcium transients with GCaMP is a key advantage for the execution and interpretation of mapping experiments.

Excitatory synaptic connections were identified by the presence of GCaMP fluorescence increase in only the TF stimulated neuron and a reproducible inward current with appropriate synaptic delay, kinetics and reversal potential in the patched cell (see Materials and methods). To separate spontaneous currents occurring during the stimulation epoch from bona fide synaptic events, we also required occurrence of synaptic responses on multiple stimulus repetitions and with a post-stimulus onset jitter of less than 14 ms. A lack of connection was defined as failure to reach these criteria following the presence of a GCaMP6s response to light stimulation in a potential presynaptic neuron.

In a representative experiment, we identified 43 nuclear-labeled cells in a single axial plane (Figure 3A), of which 35 cells yielded calcium transients in response to TF stimulation (Figure 3B). Three of these 35 cells elicited reproducible postsynaptic responses in the recorded neuron when photostimulated (Figure 3C), exhibiting multiple currents consistent with the production of trains of presynaptic action potentials. Every connection detected was associated with an unequivocal calcium transient in only the stimulated neuron (Figure 3B). In repeated experiments (seven neurons from five animals), another example of which is shown in Figure 3—figure supplement 1, the average rate of detecting a calcium response to TF stimulation of a cell expressing the ChR2-Kv-P2A-H2B-mRuby2 bicistronic construct was 80.00 ± 2.60%. Given that the GCaMP6s is expressed by a separate AAV construct and coinfection is not necessarily 100%, the 'nonresponsive' cells may express insufficient GCaMP for detection of single action potentials. Indeed, we identified three reproducible postsynaptic events (out of 316 presynaptic stimulations) that could not be correlated with a calcium transient, perhaps again due to lower GCaMP expression in those neurons. Overall, the average connectivity rate was 10.27 ± 2.60% (27 connections out of 252 cells showing a calcium transient to photostimulation), which was not significantly different from our own results using paired recording (7 connections out of 115 cells; Fisher’s exact test, p=0.18).

Figure 3—figure supplement 1. — (A) In acute slices expressing GCaMP6s (green) and targeted ChR2-P2A-H2B-mRuby2 (magenta), cells were identified for photostimulation based on nuclear mRuby2 fluorescence (yellow circles). A cell was patched and dye-filled (triangle) and assessed for postsynaptic currents as surrounding cells were stimulated (1.40 mW/µm² incident power) and GCaMP fluorescence was simultaneously recorded. Scale bar = 100 µm. (B) Changes in GCaMP fluorescence over the experimental timecourse for each cell identified in (A); each cell was stimulated sequentially with ~2 s between cells. Signals are the average of four trials. In this experiment, 35 out of 43 cells yielded calcium transients in response to optical stimulation; overall the average probability of detecting an induced calcium response was 80%. (C) Calcium (blue) responses for a subset of cells reacting to TF stimulation; four trials are overlaid in light blue, with the average in dark blue. Red lines in the calcium traces indicate the onset of stimulation for each cell. The recorded currents for the four trials (black) in the patched and putatively postsynaptic cell are shown expanded below each calcium trace; the shaded red region indicates the 150 ms stimulation epoch. Three cells showed a calcium response to TF stimulation and triggered postsynaptic currents (fast onset kinetics but delayed with respect to the TF stimulation) in the patched cell. Five representative cells showing calcium transients but no reproducible postsynaptic currents are also shown. For the displayed three connected cells and the five unconnected cells, the cell numbers, spatial locations and full calcium traces are marked with plus signs and minus signs, respectively, in panels (A) and (B). Across multiple experiments, the average connection probability was 10% (27 connections out of 252 responsive cells).

**DOI:** http://dx.doi.org/10.7554/eLife.14193.012

For comparison, we executed similar GCaMP6s-monitored mapping experiments (three neurons from two animals) using nontargeted ChR2 and the same average stimulation power (Figure 4). Although we were able to detect putative synaptic connections, they were frequently coincident with direct current responses indicative of stimulation of the patched cell’s dendritic arbor (Figure 4C). Across the experiments, such direct responses occurred during stimulation of 28.75 ± 4.46% of the target cells within a single field of view. These direct responses were often large in amplitude and could easily obfuscate much smaller synaptic events associated with bona fide connections. Moreover, we also observed reproducible calcium transients in off-target cells when other target cells were being stimulated (note events in Figure 4B that lie off of the diagonal). These off-target responses occurred with a probability (the number of events divided by the number of target cells) of 15.17 ± 1.79% and likely result from unintentional stimulation of a ChR2-containing sensitive dendrite of one cell while intending to activate the soma of a separate cell. Consistent with this interpretation, these off-target calcium events were observed at a significantly lower frequency in experiments with the targeted opsin (5.42 ± 1.39%, p<0.01 by two sample t-test). Moreover, the average distance between a cell exhibiting an off-target calcium transient and the intended target cell was greater with nontargeted ChR2 (40.6 ± 6.4 µm versus 18.9 ± 2.4 µm; p<0.01 by two sample t-test), further suggesting that cells expressing nontargeted ChR2 were firing action potentials in response to unintended stimulation of distal dendrites. Our combination of TF and restricted ChR2 thus facilitated the mapping of local circuitry within the 300 µm surrounding a neuron without confounding signals from its dendritic arbor and with a higher throughput than that achieved with electrophysiological techniques alone.

Figure 4. — (A) In acute slices expressing GCaMP6s (green) and conventional ChR2-mRuby2, cells were identified for photostimulation based on responsiveness to widefield ChR excitation (yellow circles). A cell was patched and dye-filled (triangle) and assessed for postsynaptic currents as surrounding cells were stimulated (2.13 mW/µm² incident power) and GCaMP fluorescence was simultaneously recorded. Scale bar = 100 µm. (B) Changes in GCaMP fluorescence over the experimental timecourse for each cell identified in (A); each cell was stimulated sequentially as described in Figure 3. Signals are the average of four trials. (C) Calcium (blue) responses for a subset of cells reacting to TF stimulation; four trials are overlaid in light blue, with the average in dark blue. Red lines in the calcium traces indicate the onset of stimulation for each cell. The recorded currents for the four trials (black) in the patched and putatively postsynaptic cell are shown expanded below each calcium trace; the shaded red region indicates the 150 ms stimulation epoch. Several types of events were observed: one cell showed a synaptic event coincident with a low amplitude current from direct stimulation of the dendritic arbor of the patched cell, and one cell showed what might be a synaptic event buried within another direct stimulation current of large amplitude (red arrows). Three other cells with a calcium response to TF stimulation and coincident direct stimulation of the patched cell’s dendritic arbor are also shown, along with three cells with calcium transients but no detectable currents in the patched cell. The cell numbers, spatial locations and full calcium traces corresponding to these events are marked in panels (A) and (B).

**DOI:** http://dx.doi.org/10.7554/eLife.14193.014

Discussion

This study demonstrates that combining temporal focusing for two-photon activation of ChR2 with restriction of ChR2 expression to the soma and proximal dendrites of neurons yields a reliable method for evaluating synaptic connectivity with single-neuron resolution. The spatially restricted ChR2 expression we describe allows unmasking of synaptic connections from neurons whose somata lie close to the dendrites of the postsynaptic cell and would have been occluded by direct activation of ChR2 on the dendrite. In addition, depletion of ChR2 from axons prevents inadvertent depolarization of boutons or fibers of passage that could compromise attempts to identify the source of a synaptic event. ChR2 harboring the Kv2.1 localization motif also showed enhanced sensitivity, which is of particular use in situations where excitation power is at a premium. Together these features of the soma-restricted construct significantly enhance the ability to map synaptic connections with single-cell resolution.

The development of spatially-restricted optogenetic constructs is probably one of the most effective means for achieving single-cell resolution in local circuit mapping experiments. This restriction requires the identification of a relatively small compartment near the neuronal soma characterized by selective expression of particular proteins with known motifs responsible for their localization. The distribution of particular voltage-gated potassium channels defines such a subcellular domain along the soma and proximal dendrites (Trimmer, 2015), and the sufficiency of a short Kv2.1 C-terminal sequence for driving heterologous proteins to this region (Lim et al., 2000) made this strategy ideal for restricting ChR2 expression. The only obvious alternative is restriction to the axon initial segment (AIS) mediated by an ankyrin G binding motif present in voltage-gated sodium channels (Garrido et al., 2003). Although incorporating this motif into ChR2 drives its localization to the AIS (Grubb and Burrone, 2010; Wu et al., 2013), the resulting construct does not support light-generated action potentials under physiological conditions (Grubb and Burrone, 2010). Moreover, AIS-targeted ChR2 alters the intrinsic firing properties of retinal neurons, presumably by displacing endogenous voltage-gated sodium channels whose subcellular localization is also dependent on ankyrin G binding (Zhang et al., 2015). Therefore, the Kv2.1 targeting strategy currently remains the optimal means of concentrating ChR2 such that action potentials will not be generated by TF stimulation of dendrites >20 µm away from the soma. Indeed, this approach has been successfully used with opsins to artificially generate center-surround receptive fields in retinal ganglion cells (Wu et al., 2013). The Wu et al. (2013) study showed functional restriction of opsins to the soma and proximal dendrites by examining receptive field responses to wide (200 µm) bars of one-photon light. We have now measured the somatic targeting at a finer scale, demonstrated two-photon excitation of targeted ChR2 that is enhanced relative to normal ChR2 under the same conditions, and established the utility of targeted opsins for enhancing the resolution of local connectivity maps of neural circuits.

The soma-targeted ChR2 could be combined with many of the techniques for two-photon optical control of neuronal activity previously developed for caged neurotransmitters or optogenetic probes. The relative merit of the optical activation method depends on the axial resolution, temporal precision required and the number of neurons to be stimulated simultaneously in a given experimental paradigm. For example, rapid scanning of a diffraction-limited two-photon excitation spot in a pattern on the soma can generate action potentials (Packer et al., 2012; Prakash et al., 2012; Rickgauer and Tank, 2009) and combination with spatial light modulators allows for simultaneous excitation of neuronal ensembles (Packer et al., 2012, 2015). The temporal resolution of this rapid scanning approach, however, is limited by the time required to scan along the cell body. Scanless activation of untargeted ChR in brain slices using TF, alone or in combination with spatial light modulators, is capable of generating action potentials in hippocampal pyramidal neurons within 1–3 ms of light onset (Andrasfalvy et al., 2010) and in cortical neurons in less than 10 ms (Papagiakoumou et al., 2010). We did not take full advantage of the temporal precision capability of TF to fire action potentials in our current study, instead focusing on a screening method that would identify connections without optimizing the amount of power that would fire each potential presynaptic neuron with minimal latency. We therefore chose longer pulses at a power sufficient to fire most neurons and generate trains of action potentials, which would elicit stronger signals with calcium indicators. For experiments requiring temporal precision, the minimization of action potential latency requires optimization of excitation area and laser power (Papagiakoumou et al., 2008). Because power is also the limiting factor in extending the area of two-photon activation to large numbers of neurons with spatial light modulators, the reduced power required to bring soma-targeted opsins to threshold would be an asset to such experiments.

The optimal performance of our method relies on sufficient co-expression of an opsin, a marker for opsin expression, and a genetically encoded calcium sensor. For GCaMP6s and similar sensors, there appears to be a balance between sufficient expression to detect single action potentials and excessive levels that lead to a lack of responsiveness (Chen et al., 2013; Packer et al., 2015; Tian et al., 2009). Targeting this expression window can be complicated, particularly when simultaneously trying to achieve high opsin levels using a separate AAV construct. Indeed, heterogeneity in GCaMP expression levels between neurons with viral infection may be partially responsible for the 20% of neurons in which we did not detect a change in GCaMP fluorescence with TF stimulation. We have also explored the co-expression of GCaMP6s as the second member of a single P2A-mediated bicistronic construct, but the GCaMP6s expression level was lower than desired, resulting in inadequate detection of action potentials (data not shown). Future development of these techniques will therefore benefit from bypassing viral systems and instead expressing the calcium sensor or the opsin in transgenic animals. The enhanced sensitivity of the targeted ChR2 should alleviate the concern that there would be sufficient opsin expression from a single genetic locus for activation of neurons with two-photon excitation.

There are several opportunities for future extension of this method. The somatic targeting approach could also be exploited in the context of other opsins such as C1V1, which has recently been leveraged for two-photon stimulation of neural circuits in acute slices and in vivo (Packer et al., 2012, 2015; Prakash et al., 2012; Rickgauer et al., 2014). Other molecules which may be good candidates for somatic targeting include the red-shifted ReaChR for its sensitivity (Lin et al., 2013) and Chronos for its rapid temporal characteristics (Klapoetke et al., 2014). Moreover, the somatic restriction of membrane-bound genetically encoded voltage sensors could dramatically reduce background associated with the neuropil and facilitate an all-optical version of our current method. Because the Kv2.1 targeting sequence is unable to restrict the distribution of the single-pass membrane protein CD8, however, there may be certain structural constraints on the effectiveness of the motif (Lim et al., 2000). We suspect that at least for the opsins, which are all seven transmembrane domain proteins likely to have a similar structure, the Kv2.1 motif will be sufficient to achieve somatic localization.

In summary, our combination of TF and soma-restricted ChR2 enables functional connectivity mapping and is straightforward and easy to implement with standard two-photon microscopes. Furthermore, these techniques could also be used in vivo, where the enhanced resolution of the targeted ChR2 makes it especially attractive for selective cell stimulation in behavioral paradigms. These enhancements to probing brain microcircuitry through optical stimulation promise to reveal much about nervous system function and how it might be modified by experience and perturbed in animal models of neurologic or psychiatric disease.

Materials and methods

Construct generation

The vector pAAV-hSyn-hChR2(H134R)-EYFP (Addgene plasmid 26973) served as the backbone for generating certain modified constructs. The 'proximal restriction and clustering signal' (Lim et al., 2000) of the Kv2.1 voltage-gated potassium channel (QSQPILNTKEMAPQSKPPEELEMSSMPSPVAPLPARTEGVIDMRSMSSIDSFISCATDFPEATRF), codon optimized for mouse, was generated by automated gene synthesis (Integrated DNA Technologies, Coralville, IA) and amplified by PCR using primers CGGCATGGACGAGCTGTACAAGCAGTCCCAGCCTATTCTGAAC and TGATATCGAATTCTTACTTAAACCGCGTAGCCTCTGG. The resulting product was inserted into the BsrGI site at the C-terminus of the ChR2-EYFP fusion protein sequence using the Gibson Assembly kit (New England Biolabs, Ipswich, MA). For mapping experiments using nontargeted hChR2 with coincident visualization of GCaMP6s, the EYFP-coding sequence between the PshAI and BsrGI sites was replaced with the sequence of mRuby2.

To better visualize cells for stimulation during mapping experiments, we generated a bicistronic AAV construct consisting of hChR2 followed immediately by the Kv2.1 targeting sequence, a P2A ribosomal skipping sequence, and a histone 2B-mRuby2 fusion protein. The Kv2.1 sequence was amplified with primers ATCGAGGTCGAGACTCTCGTCGAAGACGAAGCCGAGGCCGGAGCCGTGCCAGCGGCCGCCACCCAGTCCCAGCCTATTCT and ACGTCTCCTGCTTGCTTTAACAGAGAGAAGTTCGTGGCTCCGGATCCAAACCGCGTAGCCTCTGG, histone 2B was amplified from Addgene plasmid 11680 with primers CTGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCTGGTTCTATGCCAGAGCCAGCGAAG and TCGGCTTCGTCTTCGACGAGCATGGTGGCGACCGGTG, and mRuby2 was amplified from Addgene plasmid 40260 with primers CTCGTCGAAGACGAAGCCGAGGCCGGAGCCGTGCCAGCGGCCGCCACCATGGTGTCTAAGGGCGAA and GATAAGCTTGATATCGTTACTTGTACAGCTCG. The amplified products were incorporated into an EcoRI-PshAI fragment from Addgene plasmid 26973 by Gibson Assembly; the P2A sequence was reconstituted from the primers during the assembly process.

All plasmids were propagated in Stbl3 cells (Life Technologies, Grand Island, NY) and made into recombinant adeno-associated viral (AAV) particles of serotype 1 by the Penn Vector Core (Philadelphia, PA).

Animals and viral injections

All animal work was conducted according to the Guide for the Care and Use of Laboratory Animals from National Institute of Health. C57BL6/J mice were maintained on a 12 hr light-dark cycle with ad libitum access to food and water. Under isofluorane anesthesia, P21-P25 mice were injected with AAV particles (800 nl containing ~1 × 10¹³ genome copy units/ml) at 9.2 nl per 10 s through a pipette positioned 500 µm beneath the surface of the somatosensory cortex and attached to a Nanoject II microinjector. For mapping experiments with nuclear labeling and verification of action potential generation by calcium influx, full strength AAV1 encoding ChR2-Kv2.1-P2A-H2B-mRuby2 (5 × 10¹² units/ml) was coinjected with a diluted concentration of AAV1 GCaMP6s (final concentration = 1.4 × 10¹²units/ml); diluted GCaMP6s reportedly results in nuclear exclusion of the calcium indicator and lower toxicity (Packer et al., 2015). For all data reported here, animals receiving targeted or untargeted ChR2 viruses were all examined between 3 and 4 weeks after injection. Somatic restriction was also observed in animals 5 weeks after injection.

Dissociated cortical neurons

Neurons were prepared by dissecting the corticies from postnatal day 1 animals and digesting for 30 min at 37°C in Earle’s Balanced Salt Solution supplemented with 1.5 mM MgSO₄, 1 mM CaCl₂, and 8.3 units/ml papain (Worthington Biochemical, Lakewood, NJ) under 95%O₂/5%CO₂. Just prior to plating, cells were transfected with 1 µg DNA per 2.5 × 10⁶ cells using the Amaxa nucleofector system. Neurons were then seeded onto laminin-coated coverslips containing a feeder layer of astrocytes prepared as previously described (McCarthy and de Vellis, 1980). Cells were maintained in neuronal growth media (Neurobasal (Life Technologies) supplemented with 5 µg/ml insulin, 110 µg/ml sodium pyruvate, 100 units/ml penicillin, 100 µg/ml streptomycin, 40 ng/ml thyroxine, 292 µg/ml L-glutamine, 5 µg/ml N-acetyl-L-cysteine, 100 µg/ml BSA, 100 µg/ml transferrin, 16 µg/ml putrescine, 60 ng/ml progesterone, 40 ng/ml sodium selenite, 50 ng/ml BDNF, and 5 ng/ml forskolin) for ~10 days before use in experiments.

Slice preparation and electrophysiology

Three weeks after viral injection, 300–400 µm slices were prepared in ice-cold cutting solution containing (in mM): 124 choline chloride, 26 NaHCO₃, 2.5 KCl, 3.3 MgCl₂, 1.2 NaH₂PO₄, 1 glucose and 0.5 CaCl₂. After cutting, slices were allowed to recover for 30 min at 32°C in artificial CSF containing (in mM): 124 NaCl, 26 NaHCO₃, 3 KCl, 1.25 NaH₂PO₄, 20 glucose, 1 MgCl₂, 2 CaCl₂, 5 sodium ascorbate, 3 sodium pyruvate and 2 thiourea. All solutions were maintained under constant 95%O₂/5%CO₂. Whole-cell recordings were made through 4-7 MΩ pipettes, filled with intracellular solution containing (in mM): 145 potassium gluconate, 5 NaCl, 10 HEPES, 0.5 EGTA, 4 MgATP, 0.3 NaGTP, 0.02 Alexa Fluor 488 or 594 hydrazide. In some experiments, the intracellular solution was supplemented with 0.2% biocytin. All recordings were collected using Axon Multiclamp 700B amplifiers and Digidata 1440A digitizers (Molecular Devices, Sunnyvale, CA) at 10 kHz, controlled by Clampex software.

Optical setup

Our customized optical setup was based on a AxioImager Z1 platform (Zeiss, Thornwood, NY) fitted with an Ultima dual path scan head containing two pairs of galvanometric mirrors (Bruker, Middleton, WI) for separate control of imaging and stimulating optical pathways (Figure 1—figure supplement 1). The imaging pathway used a Chameleon Ti:sapphire laser tuned to 920 nm (Coherent, Santa Clara, CA). The stimulation pathway employed a MaiTai DeepSee Ti:sapphire laser tuned to 880 nm (SpectraPhysics, Santa Clara, CA). The spectral broadening necessary for temporal focusing was achieved on this path by placing a 300 line/mm diffraction grating (Thorlabs, Newton, NJ) approximately 1 m away from the galvanometric mirrors; the laser spot on the grating was imaged onto the plane between the mirrors using a 500 mm focal length lens (Thorlabs) placed 500 mm away from the grating along the path of the first diffraction order from the grating. The stimulation laser was recombined with the imaging laser in the scan head using a 900 nm long-pass dichroic mirror (Chroma, Bellows Falls, VT). The power applied by either laser was controlled with a separate Pockels cell (Conoptics, Danbury, CT).

Confocal microscopy

Three weeks after viral injection, animals were transcardially perfused with saline followed by 4% paraformaldehyde. Brains were removed, cryoprotected in 30% sucrose, and sectioned at 50 µm intervals on a sliding microtome. Sections were stained for ChR2-EYFP fusion proteins with a rabbit antibody raised against GFP followed by an Alexa 488-labeled secondary antibody (Thermo Fisher Scientific, Waltham, MA). Sections were mounted in Prolong Gold (Thermo Fisher Scientific) and images were collected on a Zeiss 780 confocal microscope using 10× air- and 40× oil-immersion objectives. Acquisition settings for laser power and PMT voltages were kept constant between nontargeted and targeted ChR2 samples.

Stimulation and resolution studies

Temporal focusing stimulation was controlled by Prairie View 5.0 software (Bruker). Standard stimulation protocols used 150 ms pulses, except as noted in the text. Stimulation powers were measured after the objective, and ranged from 15 to 285 mW; assuming our spot size to be at least 10 µm in diameter, we are using powers of no greater than 0.2 to 3.6 mW/µm². This calculation assumes a completely flat excitation profile; in reality, there is some spread in the axial dimension and thus, the power density is probably even lower. Cells filled with fluorescent Alexa dyes were visualized by two-photon microscopy and recorded in whole-cell voltage or current clamp as the temporal focusing spot was moved to different axial planes, different cell bodies or particular spots along dendrites. For each dendrite examined, the temporal focusing-induced current was plotted as a function of distance from the cell soma; the resulting curves were averaged for each construct to generate the traces shown in Figure 1C. All responses in Figure 1 were verified to exhibit kinetics associated with direct stimulation of the patched cells: that is, exhibiting onset and offset precisely locked to the start and end of the optical stimulation. Stimulation powers were chosen separately for each cell by identifying the minimum power that evoked action potentials continually across ten trials. Resolution of spike generation was measured by moving the stimulation spot at 9 µm intervals laterally or 10 µm axially above or below the cell soma and recording the proportion of trials out of 10 leading to an action potential. Data were collected from cells until a minimum of ten cells was obtained for following ChR2 expression along dendrites; given a standard deviation of ~11% for these measurements, our sample size should be able to detect a 15% difference between groups with a power of 0.8. Data for other measures were collected in parallel, and no data were ever excluded even if a recording failed prior to collecting the dendrite expression results.

Calcium imaging and connection analysis

Images of 512 × 512 pixels were simultaneously collected at 1.5 Hz using a raster scanning galvonometric system. Each stimulation epoch was timed to coincide with the onset of every other imaging frame, that is, the first spot was stimulated at the onset of the second frame, the second spot was stimulated 1.26 s later at the onset of frame 4, and so on. In this manner, a calcium peak in any frame could be associated with stimulation of a particular point in the field. During each stimulation epoch, microscope PMTs were protected from saturating fluorescence signals by Uniblitz shutters (Vincent Associates, Rochester, NY). Because this resulted in a lack of signal in the first 150 ms of every other frame as the top portion of the microscope field was scanned by the imaging laser, all stimulation points and calcium data collection were confined to a ~200 × 300 µm region of the microscope field. First-pass regions of interest (ROIs) were based on the stimulation points defined by labeled nuclei; frames containing significant calcium transients were then used to manually refine each ROI to define the soma of a responsive cell. For each ROI, the change in fluorescence relative to baseline (△F/F) was computed based on a baseline period of 10 frames prior to the onset of any stimulation. Significant calcium transients were defined as events of greater than three standard deviations above the mean for a duration of at least 2 frames. Cells with baseline GCaMP6s signals of more than 2 standard deviations above the mean in a given experiment were excluded from analysis, as GCaMP overexpression can lead to aberrant responsiveness (Chen et al., 2013; Packer et al., 2015; Tian et al., 2009). A synaptic connection was scored if the following criteria were met: (1) a latency of at least 2 ms from stimulus onset, (2) occurring in at least three out of four trials, (3) a jitter of less than 14 ms, (4) a rise time from 10% of peak to 90% of peak of less than 10 ms, and (5) the presence of a calcium transient in the photostimulated cell. Detected connections were confirmed to be excitatory in nature by altering the holding potential of the patched cell to demonstrate a reversal potential of ~0 mV. Connectivity rate was defined as then number of pairs with calcium transients in the presynaptic neuron and postsynaptic currents divided by the number of presynaptic neurons with calcium transients stimulated.

Acknowledgements

We would like to thank Alipasha Vaziri for advice on temporal focusing, Jason Christie for consultations on implementing temporal focusing on our specific two-photon setup, David Whitney and Dan Wilson for valuable conversations regarding calcium imaging, David Fitzpatrick for comments on the manuscript, and Asnel Joseph, Nowrin Ahmed, and Laura Conatser for performing stereotaxic AAV injections. GCaMP6s is courtesy of V Jayaraman, R Kerr, D Kim, L Looger, and K Svoboda from the GENIE Project at the Janelia Farm campus of the Howard Hughes Medical Institute. This work was supported by the Max Planck Florida Institute.

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Funding Information

This paper was supported by the following grant:

Max Planck Florida Institute to Christopher A Baker, Yishai M Elyada, Andres Parra-Martin, M McLean Bolton.

Additional information

Competing interests

The authors declare that no competing interests exist.

Author contributions

CAB, Conceived the study, Designed and built the optical setup, Engineered DNA constructs and performed temporal focusing experiments, Analyzed data, Wrote the manuscript.

YME, Designed and built the optical setup, Drafting or revising the article, Contributed unpublished essential data or reagents.

AP, Determined intrinsic electrophysiological properties and conducted paired recording experiments, Analyzed data.

MMcLB, Conceived the study, Analyzed data, Wrote the manuscript.

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, and all animals were handled according to protocols approved by the Institutional Animal Care and Use Committee of the Max Planck Florida Institute for Neuroscience.

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eLife. 2016 Aug 15;5:e14193. doi: 10.7554/eLife.14193.015

Decision letter

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[Editors’ note: a previous version of this study was rejected after peer review, but the authors submitted for reconsideration. The first decision letter after peer review is shown below.]

Thank you for choosing to send your work entitled "Single-cell resolution circuit mapping with temporal-focused excitation of soma-targeted channelrhodopsin" for consideration at eLife. Your full submission has been evaluated by Eve Marder as the Senior editor and three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the decision was reached after discussions between the reviewers. Based on our discussions and the individual reviews below, we regret to inform you that the work does not meet the standard for publication in eLife.

While the reviewers agreed that the strategy you propose is elegant and potentially very powerful, and could have a major impact on the field of neuroscience, unfortunately they also expressed serious concerns regarding the details of the experiments and the presentation of the results, which dampened enthusiasm for the manuscript. In particular, the section of the manuscript on circuit mapping was felt to be especially weak and would require a major series of additional experiments. As you probably know, eLife has a policy of not asking for significant new experiments as part of a revision, thus resulting in the present decision.

Reviewer #1:

In this paper, the authors describe an optogenetics method to reach optogenetics activation with single cell resolution. The approach combines the use of temporal focusing with targeted opsins, which restrict the chanelrhodopsin expression to the soma and the apical dendrites. The development of somatic opsins is one of the most promising ways of reaching a true cellular resolution with optogenetics and will surely strongly impact the neuroscience community.

However, in my opinion, the manuscript cannot be published in the present form and requires significant major modifications.

Introduction:

References reporting optical methods using two-photon excitation, diffractive optics and temporal focusing for optogenetics are not extensively cited. Also a state of the art (including eventual references) on alternative approaches to achieve somatic ChR2 targeting will help in appreciating the novelty of the paper. The decision of using Kv2.1 voltage gated potassium channel to confine ChR2 targeting should be discussed more extensively.

Results and Discussion:

Figure 1 (left): The meaning of this figure is not clear. It seems that the authors want here to characterize the optical resolution of the system. If this is the case, lateral resolution will be better characterized by performing a lateral displacement of the excitation spot along the x and y direction and plotting the corresponding curves (similarly as was done in figure 1, right panel, for axial resolution). The 3D image is very confusing and not necessary.

Figure 2—figure supplement 2: Cross sections through the images are needed in order to appreciate the values for lateral and axial FWHM.

Figure 2A: The image showing the Alexa 594 distribution (central bottom panel) has a very reduced fluorescence spreading with respect to the central top one: this difference is not justified as the spreading should be comparable in the two cells: authors should probably choose a better example.

Figure 2D: The experiment on acute slice has been done only once: this is not enough to support their conclusion more statistics is needed. They should be able to derive for acute slice a figure similar to Figure 2B and C.

They need to discuss the effect of the planarity of the dendrites in the experiments: the excitation spot has been moved laterally along the objective focal plane, if the dendritic process is axially tilted this could also induce a decrease in the current (more statistic will enables removing this ambiguity).

In order to compare data from non targeted and targeted cells, authors should comment on the time they wait after injections in the two cases. Is this comparable? How long the somatic targeting stays somatic? Is there a critical time window after which the somatic ChR2 starts spreading along dendritic processes? Scale bar should be indicated in the bottom image. Why for the targeted ChR2, data have been taken with larger step?

Figure 2F: The authors should better explain how they obtained this figure; how do they define the threshold?

Figure 2G: In the caption they write "each position in a map was stimulated with the minimum power that reliably evoked action potential when stimulation was applied to the soma": they should better quantify the meaning of "reliably evoked action potential". Stimulation protocol (pulse duration, pulse frequency) should be indicated in the caption for all the experiments.

Figure 2—figure supplement 1: Not needed.

Figure 3: The data and procedure reported in this figures needed to be better presented and explained.

A picture showing the GCamp6 fluorescence before photostimulation is needed to visualize the distribution of the cells in rest condition.

It is not clear if the cell dye-filled and imaged in A is a ChR2 positive cell. If this is the case, authors need to show the current when the photostimulation spot is placed on the cell. The experiment should be repeated more than a single time to be convincing.

The construct used in Figure 3 uses ChR2 directly linked to GCamp6: this is a very powerful idea and should be better highlighted.

Results section: "[…] owing to the lower efficiency of spike generation by ChR2 in the absence of TF […]" this sentence is wrong. TF does not increase the efficiency of ChR2 excitation but only reduce the out of focus contribution, thus improving axial resolution.

"[…] and a reproducible current with appropriate synaptic delay and kinetics […]" this sentence is very vague, authors should define and quantify what is an "appropriate synaptic delay and kinetics".

The discussion on the biological results of in Figure 3 should be toned down. The paper is a methodological paper with interesting results and does not need in my opinion a biological conclusion that is not supported by enough data.

Reviewer #2:

The authors created a new construct that localizes ChR2 to the soma and proximal dendrites of neurons. When combined with two-photon beam shaping methods (e.g. temporal focusing), this should improve the ability to target and stimulate individual densely packed neurons without concurrently activating their neighbors.

While the new construct may alleviate some of the concerns typically associated with optical mapping of connectivity (i.e. the inability to precisely stimulate only neurons of choice), the data presented in this manuscript are far too preliminary to make an impact in the field of circuit mapping.

Detailed comments:

Figure 1: Much more quantification is needed. The important variable for circuit mapping (Figure 3) is whether or not a spike is elicited, rather than the inward current. The authors should determine on what fraction of trials a spike is elicited for each power, for each location. Currently only single-trial raw current-clamp data is shown in Figure 1, but some quantification of this is required, for example:

For the final power chosen, for each neuron, what fraction of trials led to a spike when the spot was directly on the soma, and what fraction of trials led to a spike when the spot was directly, vertically, above the neuron (i.e. position iv), which seems to be the most vulnerable position for eliciting unwanted action potentials?

What was the final power used for the example shown? 61mW is on the threshold of activating the neuron soma directly (position iii), and 89mW (the next power tested) is on the threshold of activating the neuron when the beam is not directed to the soma (position iv).

As far as I can make out, the authors go on to change the protocol later in the paper (Figure 3, "circuit mapping"), using 150ms long pulses in order to generate trains of action potentials. However, all of the analysis in Figure 1 needs to be redone with these experimental parameters, since longer stimulation pulses will increase the probability of unwanted spikes away from the location of light stimulation.

What is the latency to action potential for each of the laser powers?

Figure 2: In panel 2D, the authors should show an example of a "targeted" neuron (i.e. ChR2 localized to the soma), whilst stimulating at points along the apical dendrite at the same density as that shown for the "non-targeted" neuron. Also, the current elicited in the targeted neuron is here lower than the current elicited for the non-targeted neuron, which contradicts panel E, and is not "representative" – what was the stimulation power used in the two cases?

In panel 2G, the interesting variable is the average number of spikes elicited in current clamp and these data would have been more valuable.

Figure 3: The image quality needs to be refined, and some of the somata are poorly defined. This applies particularly to the cells that are assumed to be connected.

The voltage-clamp traces in panel 3C are single trial data. The authors should show multiple traces for each connection to convince the reader that a true connection is present, rather than an EPSC which happens to coincide with light stimulation.

The authors should quantify the calcium signals in all the neurons in the imaged population when a single neuron has been targeted for stimulation (beyond what's shown in Video 1 & 2, which are not informative). Crucially, the authors must show unambiguously that there was only one neuron active on each stimulation trial.

Reviewer #3:

The authors present a novel combination of two known methods, light shaping and opsin targeting, for the purpose of mapping synaptic connections in vitro. This is in principle a very elegant approach for improving the spatial precision of optogenetic activation, currently a key limitation in the field. However, the manuscript has a rather preliminary flavor (several of the key observations appear to be n = 1). The authors are in the position to provide a major advance here by performing a detailed quantification of how accurate and reliable their method is, using ground truth calibrations. For example, the authors have not quantified how accurate their method is with any paired recordings to prove the connections they find are real. They only state that the average connectivity is similar to that in other experiments in which pairs were directly recorded. Most importantly, the lack of detailed quantification (with mean, SD, and N) needs to be addressed prior to publication.

Major comments:

1) There are major details missing in Figure 1. What is the mean action potential reliability and resolution, i.e. the grand average result of Figure 1A across all neurons? What powers were typically used for AP generation at the soma in these experimental conditions? What are the max currents observed? Please provide mean, SD, and N. Note that the figure was not created with the construct that was ultimately used, which is a weakness.

2) How many cells were used to generate the data in Figure 2—figure supplement 2? It appears that some of the differences are statistically borderline and without complete data including the sample size it is difficult to determine the reliability of this result. Also, how did the authors determine the number of significant digits to include?

3) In describing Figure 3, the authors mention that 3 photostimulation trials are performed at each location. Could the authors please show raw trials, perhaps in a lighter shade behind the average, to indicate the reliability of observed connections?

4) Figure 3 uses a different stimulation duration that rest of paper – the photostimulation time has been increased to 150 ms for Figure 3. As this value doesn't match the previous calibrations, it is very difficult to use the data in Figure 2 to calibrate Figure 3. How does the longer duration affect spatial resolution, action potential threshold, etc.?

5) Many of the calcium imaging transients in Figure 3 are quite large, and sometimes double-peaked when there is only one EPSC observed (Figure 3C, bottom row, red square). How do the authors explain the discrepancy between the fact that these long photostimulations (150 ms!) may very well induce more than one action potential, but only one post-synaptic response is observed? Many cortical synapses may depress, but not sufficiently to explain these observations.

6) What is the cutoff for a connection and how reliable is this? For example, in the bottom row of Figure 3C, fourth from the right, there is a large calcium transient and some tiny EPSCs – could these be a weak connection?

7) How often do the authors observe failures to confirm pre-synaptic action potential generation with imaging? They only say "occasionally". Excluding these from analysis could heavily bias estimation of connectivity rates!

8) In Figure 3, the authors photostimulate 192 different locations in a grid-like fashion. They don't aim to zap neurons directly, but rather by shooting at many locations, they hope to hit some neurons by chance. A quick segmentation of the image to find neurons and shoot them directly would improve accuracy, reliability, and potentially even be more efficient! Why do the authors not target neurons initially?

9) Have the authors repeated the experiment shown in Figure 3 more than once? If so, please present some grand average data.

10) Losonczy et al. 2010, cited by the authors, shows effective activation of axons. How can the authors be sure that is not occurring here? Can they provide some presynaptic patch confirmations of any of the connections they see?

[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]

Thank you for submitting your article "Single-cell resolution circuit mapping with temporal-focused excitation of soma-targeted channelrhodopsin" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Eve Marder as the Senior Editor.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. We hope you will be able to submit the revised version within two months, so please let us know if you have any questions first.

Summary:

The development of somatically targeted opsins is one of the most promising ways of achieving true cellular resolution with optogenetics and will surely have a strong impact the neuroscience community. The authors have considerably improved the quality of the manuscript by adding more data and statistical analysis. There is a strong case now for arguing that this new construct is better suited for mapping connectivity in the circuit using targeted optogenetic stimulation. However a few points of the paper still need additional work before the manuscript is ready to proceed towards publication. We encourage the authors to proceed with these final experiments as a matter of urgency, as this is a highly competitive field.

Essential revisions:

1) The power levels used in the different experiments are often missing, and this information is crucial to appreciate the spatial resolution achieved in the experiments (e.g. how far are the powers used from the saturation value?). The powers used to evoke a single AP are rather high and latency and jittering are rather long compared to what has been reported in the literature. This point is particularly weak considering that in several parts of the manuscript the authors insist on the "enhanced sensitivity "of the targeted opsin. Overall this implies that the opsins (somatic or not) used in this experiments are not very efficient and may not be suitable for experiments requiring e.g. multi-spot stimulation. Many datapoints e.g. the ones showing cellular resolution, or the connectivity experiments, are only performed using the targeted opsins and it is difficult to appreciate their importance if one can't compare the same experiments performed with the non-targeted version.

2) In order to showcase the advantage of the 'new targeted' construct, it is crucial to include the axial and lateral profile of spike probability also for the 'non-targeted' construct in Figure 2C, D. Please add this quantification to existing panels in this figure. State the power at which these curves were obtained.

3) Figure 1B: line scans to demonstrate somatic targeting are all done along dendritic processes, while no information or data are provided to show the expression confinement along axons. This would be helpful.

4) Figure 1D: "each pixel in the map show the direct current" are the authors plotting the peak current here? Moreover from this map it is difficult to understand the depolarization achieved. The same experiment performed in current clamp would allow us to learn about the spike probability for spot placed out of the target, which ultimately is the key elements to support the necessity of the somatic opsin for the connectivity experiments in Figure 3 or to appreciate the enhanced spatial resolution (see next comment).

5) Figure 3 is nice. The quantification of these connectivity mapping experiments could be included in this figure rather than only in the Results section of the text. For completion, please add an additional example of another such slice experiment in an extra supplementary figure. Also, the information on the stimulation protocol used here is very vague: "each cell was stimulated in series with 2 seconds between stimuli": how many stimuli? What power did they use? How confined is the response if experiments as the ones showed in Figure 1 C-D are done using this protocol? How do these results compare if similar experiments are done with a non-somatic opsin?

6) In the discussion the authors justify the use of high power and long photostimulation power:

"We did not take full advantage of the temporal precision capability of TF to fire action potentials in our current study, instead focusing on a screening method that would identify connections without optimizing the amount of power that would fire each potential presynaptic neuron with minimal latency. We therefore chose longer pulses at a power sufficient to fire most neurons and generate trains of action potentials, which would elicit stronger signals with calcium indicators. For experiments requiring temporal precision, the minimization of action potential latency requires optimization of excitation area and laser power"

This paper should convince us about the use of a new optogenetic construct, and (as discussed above) a more detailed characterization of the opsin showing the photostimulation area and laser power that enables AP generation with a temporal resolution and precision comparable to what has been achieved in the literature is important and should be carried out.

7) The sentence "Furthermore, these techniques could also be used in vivo, where the enhanced sensitivity of the targeted ChR2 makes it especially attractive" is misleading: in the paper the authors do show that the targeted version is more sensitive than the non-targeted one. But in both cases they use excitation powers much higher than what has been achieved in the literature and demonstrate performances (temporal resolution, latency and jittering) inferior to what has been achieved with ChR2 or C1V1 by other labs. This should be reworded.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Single-cell resolution circuit mapping in mouse brain with temporal-focused excitation of soma-targeted channelrhodopsin" for further consideration at eLife. Your revised article has been favorably evaluated by Eve Marder as the Senior editor, a Reviewing editor, and two reviewers.

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

1) There remain concerns about the intensity and duration of the illumination pulses used (150 ms). This suggests that the construct is not very efficient. Why were such long pulses used? Were shorter pulses used in some experiments? The authors should either demonstrate that their construct is also effective in triggering spikes when using shorter pulses, or provide a convincing justification for the use of longer pulses.

2) Please add to the Methods section some of the text that is currently a response to point 1, related to stimulation power needed to excite the cells. ("Our manuscript reports excitation powers of between 15 and 285 mW (for both types of opsins); assuming our spot size to be at least 10 μm wide and 10 μm thick, we are using powers of no greater than 0.2 to 3.8 mW/μm²). Expressing power as mW/μm² (rather than incident power in hundreds of mW) will be useful for readers.

3) 'Single-cell resolution' is advertised in the title, but is not well supported. We suggest changing the beginning of the title to 'Cellular resolution…'.

4) Please extend the comparison between your results and those of Wu et al. 2013 Plos ONE (since they originated the somatic restriction strategy).

eLife. 2016 Aug 15;5:e14193. doi: 10.7554/eLife.14193.016

Author response

[Editors’ note: the author responses to the first round of peer review follow.]

Reviewer #1:

Introduction:

References reporting optical methods using two photon excitation, diffractive optics and temporal focusing for optogenetics are not extensively cited. Also a state of the art (including eventual references) on alternative approaches to achieve somatic ChR2 targeting will help in appreciating the novelty of the paper. The decision of using Kv2.1 voltage gated potassium channel to confine ChR2 targeting should be discussed more extensively.

We have lengthened the Introduction to provide a larger context for the mapping experiments including additional citations for temporal focusing and two-photon microscopy. A separate paragraph in the Discussion has been included that provides a review of other approaches that have been taken to achieve somatic targeting, and the advantages of using Kv2.1 to achieve opsin subcellular restriction.

Results and Discussion:

Indeed, the reviewer understood our original aim and provided a better alternative to achieving this end. The original figure referenced has been omitted and replaced with lateral and axial resolution measurements in Figure 2C and 2D.

Figure 2—figure supplement 2: Cross sections through the images are needed in order to appreciate the values for lateral and axial FWHM.

Fluorescence measurements have been added, now as Figure 1—figure supplement 2. Fitting Gaussian functions to the measurements revealed the full width at half maximum to be even smaller than previously estimated.

Two different neurons have been chosen for the representation.

Experiments on acute slices were repeated for 10 cells expressing untargeted ChR2 and 13 cells expressing targeted ChR2. Representative data are presented in Figure 1B, with averages in Figure 1C. The data from cultured neurons have been dropped to make room for the new slice experiments.

We have tried to clarify that each dendrite under study was followed carefully as its depth changed within the slice, and that stimulation spots were chosen such that the temporal focusing spot contained a complete stretch (>10μm) of dendrite within a single focal plane. In addition, our inclusion of more cells should average out any remaining effects of dendrite planarity, as the reviewer suggests.

This information was not as explicit or extensive in the original version of the manuscript. It now appears in the Materials and methods section. All preparations were examined between 3 and 4 weeks post-injection. Somatic restriction has been observed at 5 weeks after injection, but we have not examined later time points.

Scale bar should be indicated in the bottom image. Why for the targeted ChR2, data have been taken with larger step?

As described above, the exact positions along dendrites were chosen in part based on planarity.For the representative example (Figure 1B), we tried to choose a pair of neurons where the stimulation locations were similarly spaced.

Figure 2F: The authors should better explain how they obtained this figure; how they define the threshold?

The threshold is defined as the minimum intensity that produced action potentials in ten out of ten consecutive trials. This is now explained at multiple points within the text.

This is the aforementioned threshold intensity for a given neuron.

Stimulation protocol (pulse duration, pulse frequency) should be indicated in the caption for all the experiments.

The captions for Figures 1–3 now contain descriptions of the stimulation protocol.

Figure 2—figure supplement 1: Not needed.

Figure 3: The data and procedure reported in this figures needed to be better presented and explained.

A picture showing the GCamp6 fluorescence before photostimulation is needed to visualize the distribution of the cells in rest condition.

This figure has been completely redone in the new manuscript. Baseline GCaMP fluorescence is shown in Figure 3A.

In the original manuscript, the cell shown was indeed ChR2-positive. In all our mapping experiments, the patched cells express ChR2 and we use their responsiveness to calibrate the photostimulation power. We have included examples of current traces found with direct stimulation of neuronal somata in Figure 1B. As for repetition of the experiment, we have indeed done this and report average statistics in the text of the Results section (seventh paragraph).

The construct used in figure 3 uses ChR2 directly linked to GCamp6, this is a very powerful idea and should be better highlighted.

We agree with the reviewer that this is a potentially powerful approach, however, in practice we found that this method actually led to decreased GCaMP responsiveness that contributed to poor signal to noise ratios and suboptimal image quality. We have not completely characterized the reasons for this but suspect that linking high opsin expression to high GCaMP expression on the same construct leads to many cells with GCaMP in their nuclei that no longer exhibit calcium transients. As a result, we turned to the approaches that other groups employ—using a dilution of the GCaMP virus—and combined this with nuclear labeling of neurons making identification easier for stimulation by temporal focusing.

We thank the reviewer for this correction. The efficiency of ChR2 excitation is quite high as indicated by its two-photon cross section. We intended to refer to the low single-channel conductance of ChR2 and suggest that when the excitation is axially confined, a rapid scanning approach or a light sculpting technique is needed to quickly activate many ChR2 molecules and drive spike generation. We have clarified this in the text and actually expanded the portion of the Results that discusses ChR2 activation by the imaging beam (fourth paragraph).

We define a bona fide postsynaptic response as exhibiting a rise time of less than 10 ms, and having a greater than 2 ms latency from the photostimulation and with less than 14 ms jitter.

These criteria are now described in the Materials and methods section (subsection “Calcium imaging and resolution studies”).

We agree, and do not desire to make a biological statement about layer 3-local excitatory connectivity. Rather, we think it is important to provide the reader with a measure of the sensitivity and reliability of this new approach for mapping synaptic connections with the current gold standard: paired patch recording. We have revised the text to clarify the rationale for inclusion of this comparison.

Reviewer #2:

[…] Figure 1: Much more quantification is needed. The important variable for circuit mapping (Figure 3) is whether or not a spike is elicited, rather than the inward current. The authors should determine on what fraction of trials a spike is elicited for each power, for each location. Currently only single-trial raw current-clamp data is shown in Figure 1, but some quantification of this is required, for example:

In response to the reviewer’s concerns we have abandoned the original figure in favor of an analysis examining spike probability for ten neurons over ten trials at axially and laterally displaced locations. These data are now reported in the current manuscript as Figure 2C and 2D.

All experiments in the paper have now been performed with the longer 150 ms stimulation protocol.

What is the latency to action potential for each of the laser powers?

The average latency for the mapping experiments was 39 ms, which is now mentioned in the text.

The new version of this experiment is presented in Figure 1B. The stimulation density is similar for both the targeted and nontargeted examples, and examining over 10 cells per group allowed us to choose truly representative cells that showcase the heightened current achieved with the targeted construct even with equivalent power (167mW in these cases).

In panel 2G, the interesting variable is the average number of spikes elicited in current clamp and these data would have been more valuable.

As mentioned previously, spike probability as a function of lateral or axial displacement is now included in Figure 2C and 2D. For our mapping experiments evaluating small synaptic currents in response to stimulation of putative presynaptic cells, however, showing a reduction in the currents evoked by direct stimulation of the dendritic arbor of the patched cell is still relevant.

Figure 3: The image quality needs to be refined, and some of the somata are poorly defined. This applies particularly to the cells that are assumed to be connected.

New images of cells are provided in Figure 3A; a fluorescent nuclear label and better GCaMP expression allowed for more defined images of all cells.

All four voltage clamp traces used to determine the presence or absence of a synaptic connection are now shown in Figure 3C.

These data are now included in Figure 3B, where the calcium signal is shown for each targeted neuron in a given experiment. Every connection is unequivocally associated with only one active neuron.

Reviewer #3:

[…] 1) There are major details missing in Figure 1. What is the mean action potential reliability and resolution, i.e. the grand average result of Figure 1A across all neurons? What powers were typically used for AP generation at the soma in these experimental conditions? What are the max currents observed? Please provide mean, SD, and N. Note that the figure was not created with the construct that was ultimately used, which is a weakness.

The resolution of spike generation in 10 cells expressing the targeted construct is now presented in Figures 2C and 2D. These measurements were determined at the threshold power for each particular cell, the distribution of which is shown in Figure 2B. The average currents at each power are shown in Figure 2A. There was no difference between targeted and untargeted cells in the average current necessary to evoke action potentials (i.e., rheobase), only a difference in the amount of stimulation power required to evoke such current.

These data are now in Figure 2—figure supplement 2, and include N for all measurements. Significant digits are determined by the measurement limits of the Clampex patch clamp software.

All raw trials are now shown in the new Figure 3C, with calcium traces overlaid in a lighter shade and electrophysiological traces displayed adjacent to each other.

All data in the paper are now derived from 150ms stimulation.

5) Many of the calcium imaging transients in figure 3 are quite large, and sometimes double-peaked when there is only one EPSC observed (Figure 3C, bottom row, red square). How do the authors explain the discrepancy between the fact that these long photostimulations (150 ms!) may very well induce more than one action potential, but only one post-synaptic response is observed? Many cortical synapses may depress, but not sufficiently to explain these observations.

Indeed, the longer stimulation protocol was used in hopes of generating trains of presynaptic action potentials. While single postsynaptic currents are observed (some neurons only generate one action potential regardless of stimulation intensity), we frequently saw large calcium transients and trains of postsynaptic currents, which are shown in the new Figure 3C.

In the original version of the manuscript, the currents described by the reviewer did not occur across multiple trials. In the new experiments reported here, we require that events occur in at least three out of four trials and with less than 14 ms jitter and a greater than 2 ms latency from the photostimulation. These criteria are now described in the Materials and methods section (subsection “Calcium imaging and connection analysis”).

The exact numbers of excluded connections from the current study are now described in the Results. For the majority of cells (5 out of 7 in the current study), every connection found could be associated with a somatic calcium transient. Out of the 36 connections identified across all cells in the present study, 3 were excluded due to lack of a somatic calcium signal.

We originally hoped that baseline GCaMP fluorescence would be sufficient to target neurons as the reviewer suggests, however, we found that many neurons with significant calcium responses to TF stimulation were impossible to identify under baseline imaging conditions. We agree with the reviewer that segmentation would be preferable, and developed a new construct in which neuronal nuclei were fluorescently labeled for easy targeting for TF stimulation. All mapping studies in the current version of the paper use this construct.

9) Have the authors repeated the experiment shown in Figure 3 more than once? If so, please present some grand average data.

The data for seven different mapping experiments are now described in the Results section (seventh paragraph).

Targeted ChR2 expression is not seen in the axons of dye-filled cultured neurons, which is consistent with the known exclusion of Kv2.1 from axons beyond the initial segment.

[Editors’ note: the author responses to the re-review follow.]

Essential revisions:

The power levels used in most experiments were calibrated for each cell to generate a single action potential; we chose this approach to compare resolution and sensitivity across multiple cells that may differ in absolute levels of opsin expression. We have made an extra effort to explicitly mention this rationale as well as the average powers chosen for each experiment; for sample mapping experiments in Figures 3 and 4, we have also included specific power levels in the figure legends.

With temporal focusing excitation distributed across a volume, the power levels for opsin excitation are expected to be higher than those used when scanning a much smaller diffraction-limited spot. In earlier papers using temporal focusing stimulation of ChR2, Andrasfalvy et al. (PNAS, 2010) reported action potential generation anywhere with 300-400 mW power at the sample plane, and Losonczy et al (Nature Neurosci., 2010) report power levels between 50 and 500 mW. Our manuscript reports excitation powers of between 15 and 285 mW (for both types of opsins); assuming our spot size to be at least 10μm wide and 10μm thick, we are using powers of no greater than 0.2 to 3.8 mW/μm². This approaches but does not surpass excitation powers of 0.45 mW/μm² reported by the Emiliani group, but differences in opsin delivery and expression may account for some of the discrepancy. Finally, Rickgauer et al. (Nature Neurosci., 2014) were able to use less than 100 mW over the entire temporal focusing disc when employing C1V1, which could be due to the enhanced optical response of that opsin relative to ChR2.

We agree with the reviewers that additional comparisons between nontargeted and targeted opsins would reveal the utility of a somatic targeting approach. We have taken this approach to heart and included new comparisons in Figure 2, and also added a new Figure 2—figure supplement 3 and Figure 4 (see below).

The manuscript now has a new version of Figure 2 with the nontargeted resolutions incorporated (see also sixth paragraph of Results). As alluded to above, the power levels for all the resolution experiments (including both Figure 1 and Figure 2) vary from cell to cell and are set to be the minimum power that generates a single action potential in ten out of ten trials. The average powers for these experiments are reported in third paragraph of Results and a mistaken power level was corrected.

We conducted similar optical stimulation experiments on axonal processes, added more detailed imaging, and included the results as Figure 1—figure supplement 3 and in the Results (fourth paragraph).

Indeed, we are plotting the peak current amplitudes in Figure 1D. This voltage clamp experiment remains an important demonstration of the extent of direct currents that overwhelm smaller synaptic events in mapping experiments, which can be seen now in the new Figure 4. We agree that the spike probability at off target locations is worthwhile information and were able to investigate this in the context of actual connectivity mapping experiments by extracting information from our previous data as well as new experiments with nontargeted opsin. When using a nontargeted construct, we found an increased probability of calcium transient generation in off-target cells when stimulating separate cells in the slice. Moreover, the average distance between an off-target activated cell and the stimulation point was greater when using nontargeted ChR2. These observations are now reported in the Results.

We appreciate the reviewer’s comments about the new figure, and have incorporated more quantification from multiple connectivity experiments into the figure legend, as requested. We have also included another example slice experiment as Figure 3—figure supplement 1. We have also tried to explain in the figure legend that each cell is stimulated sequentially at the onset of every other imaging frame (i.e., 2.4 seconds after the previous cell in the field). After going through all the cells in the field (40-80 cells), the entire protocol is repeated for three additional iterations to produce the results in the figures. We have reported the average power value across the mapping experiments seventh paragraph of Results and the specific power levels for each representative experiment within the figure legend. Finally, we appreciate the suggestion to perform mapping experiments with nontargeted ChR2, and have included an example as Figure 4. This allowed us to demonstrate unintended activation of off-target cells as well as the extent to which direct activation of the dendritic arbor occurs and can make interrogation of local connectivity problematic.

6) In the discussion the authors justify the use of high power and long photostimulation power:

"We did not take full advantage of the temporal precision capability of TF to fire action potentials in our current study, instead focusing on a screening method that would identify connections without optimizing the amount of power that would fire each potential presynaptic neuron with minimal latency. […] For experiments requiring temporal precision, the minimization of action potential latency requires optimization of excitation area and laser power"

Our goal with this manuscript was to provide a baseline system from which to base further refinements of optogenetic mapping experiments and to establish the utility of somatic opsin targeting for such an approach. We therefore focused on greater characterization of spatial resolution of the targeted ChR2 and less on temporal precision. Nevertheless, we now include data that show action potential latency consistent with that achieved in the literature can be obtained by increasing stimulation power (Figure 2—figure supplement 3). Our mapping experiments in Figures 3 and 4 also demonstrate that the advantages in spatial resolution with the targeted construct are maintained at higher stimulation powers that produce shorter latencies.

We altered the statement to focus more on spatial resolution (final paragraph of Discussion).

[Editors' note: further revisions were requested prior to acceptance, as described below.]

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

We implemented a longer stimulation protocol as this provided the greatest probability of eliciting trains of action potentials, leading to increased calcium signals in mapping experiments. In an earlier version of our manuscript, we characterized responses for both types of opsins using a shorter stimulation time (32 ms). We have now included some of these data as an additional figure supplement (Figure 2—figure supplement 3) and added appropriate commentary in the text (fourth paragraph of Results). Moreover, the ability to reduce latency to less than 10 ms with increased power (Figure 2—figure supplement 2) suggests that even shorter stimulation pulses may still be effective.

2) Please add to the Methods section some of the text that is currently a response to point 1, related to stimulation power needed to excite the cells. ("Our manuscript reports excitation powers of between 15 and 285 mW (for both types of opsins); assuming our spot size to be at least 10 μm wide and 10 μm thick, we are using powers of no greater than 0.2 to 3.8 mW/μm²). Expressing power as mW/μm² (rather than incident power in hundreds of mW) will be useful for readers.

We have now expressed all powers in the text and figures as mW/μm², and added an explanatory note to the Materials and methods section (subsection “Stimulation and resolution studies”).

3) 'Single-cell resolution' is advertised in the title, but is not well supported. We suggest changing the beginning of the title to 'Cellular resolution…'.

We have made the requested change.

4) Please extend the comparison between your results and those of Wu et al. 2013 Plos ONE (since they originated the somatic restriction strategy).

We extended the discussion of the Wu paper, mentioning their demonstration of somatic restriction by physiologic criteria (second paragraph of Discussion). As they measured the physiologic responses of cells under very different conditions than in our study, it is difficult to directly compare their estimates of opsin restriction with our own.

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PERMALINK

Cellular resolution circuit mapping with temporal-focused excitation of soma-targeted channelrhodopsin

Christopher A Baker

Yishai M Elyada

Andres Parra

M McLean Bolton

Roles

Abstract

eLife digest

Introduction

Results

Figure 1. Characterization of soma-targeted channelrhodopsin (ChR2).

Figure 1—figure supplement 1. Custom two-photon microscope setup for temporal focusing stimulation with simultaneous imaging.

Figure 1—figure supplement 2. Orthogonal projections from a z-stack of a spin-coated layer of 200 nm fluorescent beads imaged with the temporal focusing beam and collected with a CCD camera.

Figure 1—figure supplement 3. Characterization of targeted ChR2 in axonal processes.

Figure 2. Enhanced sensitivity and resolution with soma-targeted ChR2.

Figure 2—figure supplement 1. Action potentials triggered in neurons expressing nontargeted (N) or targeted (T) ChR2 in response to stepwise current injections ranging from −100 to 110 pA in 30 pA steps.

Figure 2—figure supplement 2. Action potential latency as a function of stimulation power in cells expressing targeted (red) or nontargeted (blue) ChR2.

Figure 2—figure supplement 3. Latency to first action potential with targeted and nontargeted ChR2.

Table 1.

Figure 3. Typical mapping experiment with action potential validation by GCaMP6s.

Figure 3—figure supplement 1. Second typical mapping experiment with action potential validation by GCaMP6s.

Figure 4. Typical mapping experiment using nontargeted ChR2.

Discussion

Materials and methods

Construct generation

Animals and viral injections

Dissociated cortical neurons

Slice preparation and electrophysiology

Optical setup

Confocal microscopy

Stimulation and resolution studies

Calcium imaging and connection analysis

Acknowledgements

Funding Statement

Funding Information

Additional information

Competing interests

Author contributions

Ethics

References

Decision letter

Roles

Author response

ACTIONS

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