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Simultaneous all-optical manipulation and recording of neural circuit activity with cellular resolution in vivo

Nature Methods volume 12, pages 140–146 (2015)Cite this article

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A Corrigendum to this article was published on 30 June 2015

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Abstract

We describe an all-optical strategy for simultaneously manipulating and recording the activity of multiple neurons with cellular resolution in vivo. We performed simultaneous two-photon optogenetic activation and calcium imaging by coexpression of a red-shifted opsin and a genetically encoded calcium indicator. A spatial light modulator allows tens of user-selected neurons to be targeted for spatiotemporally precise concurrent optogenetic activation, while simultaneous fast calcium imaging provides high-resolution network-wide readout of the manipulation with negligible optical cross-talk. Proof-of-principle experiments in mouse barrel cortex demonstrate interrogation of the same neuronal population during different behavioral states and targeting of neuronal ensembles based on their functional signature. This approach extends the optogenetic toolkit beyond the specificity obtained with genetic or viral approaches, enabling high-throughput, flexible and long-term optical interrogation of functionally defined neural circuits with single-cell and single-spike resolution in the mouse brain in vivo.

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Figure 1: Single-cell two-photon optogenetic photostimulation and single-action-potential readout in vivo.
Figure 2: Precise, concurrent photostimulation of multiple identified neurons in vivo.
Figure 3: Simultaneous fast calcium imaging and concurrent photostimulation of multiple neurons in vivo.
Figure 4: Dependence of network perturbations on behavioral state.
Figure 5: Targeting neurons for optogenetic manipulation based on their individual functional signatures in vivo.

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  • 06 February 2015

    In the version of this article initially published, the size of the scale bar reported in the legend of Figure 3a was incorrect. The correct size is 100 μm, not 50 μm. In addition, the volume of injected virus in the Online Methods section "Titration of calcium indicator expression" had the incorrect unit. The correct volume is 100 nl, not 100 μl. The errors have been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank M. Pachitariu, C. Stringer, S. Turaga, M. London and N. Pettit for helpful discussion, analysis routines and software; C. Wilms, C. Schmidt-Hieber, A. Roth, B. Clark, I. Bianco, S. Smith, B. Judkewitz and D. Peterka for discussion and comments on the manuscript; the staff at Bruker Corporation (formerly Prairie Technologies) for enabling customization of the microscope; M. Lochrie at the Stanford Neuroscience Gene Vector and Virus Core (grant no. P30 NS069375-01A1) for advice on use of AAVdj; and K. Deisseroth (Stanford University) for plasmids and access to AAVdj virus. This work was supported by grants from the Wellcome Trust, the Gatsby Charitable Foundation, the European Commission (Marie Curie International Incoming Fellowship grant no. 328048), the European Molecular Biology Organization, the Medical Research Council and the European Research Council.

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Authors and Affiliations

  1. Wolfson Institute for Biomedical Research, University College London, London, UK

    Adam M Packer, Lloyd E Russell, Henry W P Dalgleish & Michael Häusser

  2. Department of Neuroscience, Physiology and Pharmacology, University College London, London, UK

    Adam M Packer, Lloyd E Russell, Henry W P Dalgleish & Michael Häusser

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Contributions

A.M.P., L.E.R. and H.W.P.D. performed experiments and analyzed data. A.M.P., L.E.R., H.W.P.D. and M.H. designed the study and wrote the paper.

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Correspondence to Michael Häusser.

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Integrated supplementary information

Supplementary Figure 1 Spatial resolution of spiral two-photon photostimulation in vivo

(a) Lateral resolution of action potential photostimulation of a layer 2/3 barrel cortex neuron in vivo using a single spot generated by the SLM scanned in the 20 μm spiral pattern (using 6 mm galvos; see Methods, n = 6 neurons).

(b) Same as above for axial resolution (n = 5 neurons). The asymmetry results from the fact that as the photostimulation pattern is directed deeper than the neuron (negative values), the probability of action potential generation drops more sharply.

Supplementary Figure 2 Imaging over large fields of view does not cause photostimulation

(a) Example cell-attached patch clamp recording from one neuron. Periods of imaging are indicated by the shaded regions. Imaging conditions are indicated above the trace (schematic field of view shows a pipette and the recorded cell). Imaging was performed at 30 Hz with 920 nm excitation (see Methods) but the size of the field of view (optical zoom) was changed.

(b). There is no significant difference between spontaneous firing rate and that while imaging a 400x400 μm field of view at 50 mW power on sample (the conditions used in this paper; Friedman test, post hoc Dunn's multiple comparison test). Inset shows firing rates while imaging a 400x400 μm field of view, normalized to the recorded cells spontaneous firing rate; error bars represent SEM. Although the 920 nm light used for imaging is less than one-third as effective as the 1064 nm wavelength at stimulating the C1V1 opsin (Prakash et al 2012), sufficient current does accumulate in the neuron and surrounding local network when imaging at higher optical zooms. This type of stimulation could be used to optogenetically generate activity in localized circuits. n = 8 recorded neurons, 3 mice.

Supplementary Figure 3 Single cell two-photon raster photostimulation in vivo

(a) Top: Cell-attached patch clamp recording from a neuron in layer 2/3 of barrel cortex expressing C1V1-2A-YFP in vivo overlaid with the photostimulation pattern (pipette, magenta; YFP, green; photostimulation pattern, grid of red spots; scale bar, 10 μm.) Bottom: Raster of spike times around stimulus delivery over 10 trials, with the electrophysiological recording from trial 1. Note the time-locked precision and reliable response to photostimulation (red bar).

(b) Top: Raster of spike times around stimulus delivery for four neurons photostimulated for 40 ms. Bottom: Raster of spike times around stimulus delivery for nine neurons photostimulated for 120 ms.

(c) Lateral (top) and axial (bottom) resolution of photostimulated action potentials.

Supplementary Figure 4 Single cell two-photon raster photostimulation of an interneuron in vivo

(a) Photostimulation (120 ms grid, red bar) of a fast-spiking putative interneuron in layer 2/3 of barrel cortex in vivo (inset, two-photon targeted cell-attached patch clamp recording, scale bar 10 μm) revealed robust action potential generation despite ongoing spontaneous activity (raster).

(b) The average spike rate during photostimulation (red bar) increased sharply relative to the high background rate.

Supplementary Figure 5 Activation versus distance from nearest stimulated neuron during simultaneous activation of ten target neurons

Pooled data from multiple experiments where 10 neurons were photostimulated in layer 2/3 of barrel cortex, and the resulting activity was measured in the local network. Neurons are binned by distance from nearest stimulated neuron in 10 μm increments. The red bin at zero is comprised solely of the photostimulated neurons. Grey bins contain all non-stimulated neurons in the field of view. Open circles are individual neurons. The white line and shading indicates mean ± SD background firing rate across all non-stimulated neurons during non-stimulation periods. The curve defining the spatial resolution of single cell photostimulation from Supplementary Figure 3 is overlaid in black. Note that numerous cells at 50 μm or greater from the nearest stimulated cell are above the level of background spontaneous activity, which are not likely to have been directly photostimulated. White stars indicate significance of average spike count within a bin versus baseline (Mann-Whitney test p < 0.05 [Bonferroni corrected for 33 bins p < 0.0003]). n = 3 mice, one FOV and one 10-neuron stimulation pattern per mouse with 10 trials per repeat and ~14 repeats per mouse. Total of 672 imaged neurons, 30 of which were stimulated in 370 trials.

Supplementary Figure 6 Correlations of response to photostimulation with GCaMP6s and C1V1 expression levels

(a) Significant inverse relationship (Spearman rho = 0.40; p < 0.0001) between normalized response to photostimulation (sum of inferred spikes post-stimulation) and normalized GCaMP6s expression levels (mean brightness of region of interest containing neuron) indicates that the more strongly expressing neurons do not respond as well to the perturbation. Given the high reliability of the perturbation (Figs. 2 & 3), this is likely due to the known issue of GCaMP overexpression leading to aberrant physiology (Chen et al 2013, Tian et al 2009). The response to perturbation could thus be used to calibrate the dynamic range of the GCaMP6s signal per action potential in each neuron separately. These data are from experiments in which ten neurons were photostimulated simultaneously.

(b) There is a small but insignificant correlation (Spearman rho = 0.06, p = 0.60) between the normalized response to photostimulation and the normalized C1V1 expression level (as indicated by the mean brightness of mCherry, which is expressed in stoichiometric concentration due to the 2A peptide). These data are from experiments in which ten neurons were photostimulated simultaneously (not including the top 25% brightest GCaMP6s-expressing neurons based on a).

Supplementary Figure 7 Longitudinal stability of all-optical photostimulation and readout

(a) Imaging and selection of targets in layer 2/3 of somatosensory cortex on Day 1 of a representative experiment. White circles are the photostimulated targets (scale bar, 100 μm).

(b) The same field of view as (a), one week later.

(c) Mean ± SD calcium transient (for ten photostimulation trials) of the target cell in the dashed circle in (a) on day 1 (black) and day 8 (gray).

(d) Data from three fields of view showed the mean responses to photostimulation on day 1 and day 8 were highly correlated, implying the same responses could be observed from the same neurons one week apart. The dashed line is the unity line. The solid grey line indicates the fit with 95% confidence interval shaded in grey.

Supplementary Figure 8 Optimized GCaMP6s expression

(a) A narrow window of viral titer (rows) is crucial for sufficient and long-lasting expression over time (columns). Note lack of expression with titer that is too low (bottom row) and brightly-filled neurons with titer that is too high (top row).

(b) Quantification of the percentage of brightly-filled cells in depth-matched (~160 µm deep) fields of view at titers and time-points corresponding to the rows and columns in (a) from acute craniotomy preparations. Data from 11 mice.

(c) Example cell-matched fields of view from a chronic window preparation at 4 time-points post-injection using the 1012 genomes ml-1 titer highlighted in red in (a) and (b). Note example cells identified across 4 weeks highlighted in red.

(d) Monoexponential fit (curves) to cell-matched calcium transient decays (points) recorded at each time point (see figure key) during spontaneous activity under 0.5% isofluorane anesthesia. Transients were identified following a threshold crossing of 20% above the median value of each cell's dF/F trace. They were then normalised to the peak following this crossing. n = 2 animals (1323 cells total)

(e) Quantification of the change with time of the decay time constant (Tauoff) of the monoexponential fits shown in (d).

(f) Amplitude of cell-matched calcium transients measured at each time point. Transient threshold is shown as dotted red line.

(g) Percentage of brightly-filled neurons in analysed fields of view at each time point.

Supplementary Figure 9 Sensitivity of spike readout

(a) Confirmation of single action potential resolution in a GCaMP6s-expressing neuron obtained during two-photon targeted cell-attached patch clamp recording while imaging at 60 Hz.

(b) The calcium rise recorded from this example neuron in response to one or more action potentials shows the characteristic amplitude and time course of GCaMP6s (top traces). The inferred action potential probability from the deconvolution algorithm indicates the likelihood of a spike somewhat smeared over time.

(c) Integrating the inference over a time-window of 433 ms results in a high coefficient of determination (R2 = 0.54) of the linear fit (y = 0.63x + 0) between inferred probability of spiking and actual spiking recorded in cell-attached configuration. 433 ms resulted in a better fit versus other durations between 100 and 1000 ms. n = 4 neurons. SEM across all 250 ms epochs containing a reported number of spikes: 0 spikes n = 141; 1 spike n = 332; 2 spikes n = 236; 3 spikes n = 64; 4 spikes n = 6.

(d) Analysis confirming the ability of GCaMP6s to reliably report single spikes, based on Chen et al 2013. Hit rate and false positive rate for identification of single isolated spikes was calculated by using a template and threshold of correlation as determined using the neuron in (a). Total of 266 single spikes and 45 no spike periods from 3 neurons, not including neuron (a).

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Packer, A., Russell, L., Dalgleish, H. et al. Simultaneous all-optical manipulation and recording of neural circuit activity with cellular resolution in vivo. Nat Methods 12, 140–146 (2015). https://doi.org/10.1038/nmeth.3217

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