Two-Photon Bidirectional Control and Imaging of Neuronal Excitability with High Spatial Resolution In Vivo.

Animal Surgery

All experiments were carried out according to the guidelines of the European Communities Council Directive and approved by the Instituto Italiano di Tecnologia (IIT) Animal Health Regulatory Committee and by the National Council on Animal Care of the Italian Ministry of Health (authorization 29-2011-A, 34/2015-PR). Animals were housed in individually ventilated cages under a 12-hr light:dark cycle. A maximum of 5 animals per cage was allowed. Access to food and water was ad libitum . Experiments were performed on young-adult animals (5–16 weeks old for in vivo experiments, 4–7 weeks old for in vitro experiments, either sex). Details about animal strains and viral injections are described in the Supplemental Experimental Procedures . For in vivo experiments, mice were anesthetized with intraperitoneal urethane (16.5%, 1.65 g/kg). The scalp was removed while infiltrating all incisions with lidocaine. A chamber with a central hole (hole diameter: 4 mm) was attached with dental cement to the animal’s skull for head-fixation. A craniotomy (∼700 × 700 μm 2 ) was opened over the somatosensory (or visual cortex, in the case of experiments in Scnn mice) cortex, and the dura was carefully removed (unless otherwise stated). The location of the craniotomy was guided by the intensity of the fluorescence signal of the expressed transgene. The surface of the brain was kept moist with normal HEPES-buffered artificial cerebrospinal fluid (ACSF) (composed of 127 mM NaCl, 3.2 mM KCl, 2 mM CaCl 2 , and 10 mM HEPES [pH 7.4]). Body temperature was maintained at 37°C with a heating pad. Respiration rate, heartbeat, eyelid reflex, vibrissae movements, and reactions to tail pinching were typically monitored throughout the surgery and the experiment.

Optical Setup and Phase

Modulation for Holographic Illumination See Supplemental Experimental Procedures . In Vivo Electrophysiological Recordings Two-photon targeted juxtasomal electrophysiological recordings were performed as described in De Stasi et al. (2016) and Zucca et al. (2017) . Borosilicate glass pipettes were pulled with a resistance of 4–9 MΩ and were filled with ACSF solution mixed with Alexa Fluor 488 or 594 (20 μM). Neurons were targeted by imaging the fluorescent reporter with the two-photon microscope while monitoring the pipette electrical resistance by applying brief voltage pulses. Additional details are reported in the Supplemental Experimental Procedures . In Vivo Two-Photon Imaging and Photostimulation Two-photon imaging (λ exc = 1,050 nm) was performed to assess the expression pattern of the opsin (ChR2-eYFP or ChR2-eYFP soma targeted) and the calcium indicator (jRCaMP1a) at the same time. A reference image of the selected FOV was acquired, and shapes covering the soma of target neurons were generated by the SLM (λ exc = 920 nm) and projected at the sample. Temporal series were acquired in raster scanning configuration with the imaging beam (100 × 100 pixels; frame rate: 11 Hz; pixel dwell time: 4 μs; λ exc = 1,100 nm). Holographic photostimulation duration was 500 ms and was repeated at 0.08 Hz for 7–9 repetitions. For analysis, temporal series acquired in vivo were imported into the ImageJ/Fiji software to identify regions of interest (ROIs). For each ROI, the change in fluorescence relative to the baseline (ΔF/F 0 ) was computed as a function of time with the fluorescence baseline (F 0 ) calculated in ten frames at the beginning of the recorded session. Artifactual fluorescence signals due to holographic stimulation were removed by background subtraction ( Figure 6 B; Figure S7 ) or required blanking ( Figure 6 D). Slice Electrophysiology See Supplemental Experimental Procedures .

Data Analysis and Statistics

For juxtasomal recordings, traces were high-pass filtered (cutoff frequency: 10 Hz) and spikes were detected with a threshold criterion. The threshold value was adjusted for each recorded sweep and set >3 times the SD of the trace. For experiments in Figures 1 , 6 , and 3 and in Figures S3 and S5 , AP firing frequency was calculated in a time window Pre (window duration: 1 s), Stim (duration: 0.5 s) and Post (duration: 1.5 s) holographic stimulation over 15–20 stimulation trials. Δ AP Freq was calculated as the difference between the firing frequencies of the Stim and Pre time windows. Opsin-positive cells (for definition, see In vivo electrophysiological recordings in the Supplemental Experimental Procedures ) were considered responsive to holographic stimulation when Δ AP Freq was >1 times the firing frequency in the Pre period at stimulation power ≤ 92 mW per shape. The fraction of opsin-positive neurons responding to holographic illumination was 14/16 for Ca 2+ /calmodulin-dependent protein kinase II-positive (CaMKII + ) cells expressing ChR2 ( Figures 1 C–1E), 15/17 for layer 2/3 cells expressing ChR2 under the human synapsin promoter ( Figures 1 E and 1F), 31/33 for SST + cells expressing ChR2 ( Figure 3 ), 25/26 for PV + cells expressing ChR2 ( Figure 3 ), 19/20 for Scnn + cells expressing ChR2 ( Figure 3 ), and 21/21 for cells expressing the soma-targeted ChR2 under the human synapsin promoter ( Figure 2 ). To compute the spatial resolution, neuronal responses (quantified as Δ AP Freq) were recorded first with the stimulation shape centered on the cell body and then during successive shifts of the excitation volume in the radial (20 μm steps) and in the axial (±25 μm steps) directions. Δ AP Freq as a function of the shift was then plotted for every recorded neuron in the three conditions (radial, axial up , and axial down ) and fitted with a mono-exponential function (Δ AP Freq(x) = A ∗ exp(−l ∗ x)) ( Packer et al., 2015 ). Fitting curves with l < 0 or with values of A that were different by more than 25% compared to Δ AP Freq at position x = 0 were not considered. The spatial resolution, l 1/2 , was defined as the distance at which the evoked response (calculated from fit) was equal to A/2. For the analysis of the recordings from GtACR2 expressing neurons, see Supplemental Experimental Procedures .

Statistical Methods

All values are expressed as mean ± SEM unless otherwise stated. For each experimental group, sample size was chosen based on previous studies ( Carrillo-Reid et al., 2016 , Packer et al., 2015 , Rickgauer et al., 2014 ). No statistical methods were used to predetermine sample size. All recordings with no technical issues were included in the analysis. For N ≥ 10, a Kolmogorov-Smirnov normality test was used to test for normality. For N < 10, a Saphiro-Wilk normality test was adopted. In case of normal distribution, Student’s t test was used to calculate statistical significance when comparing two populations of data. For non-normal distributions, the non-parametric Mann-Whitney test or Wilcoxon signed-rank test (for unpaired or paired comparison, respectively) was used unless otherwise stated. When multiple (>2) populations of data were compared, one-way ANOVA with Bonferroni or Tukey’s honestly significant difference (HSD) post hoc test was used in case of Gaussian distribution. For non-normal distribution and multiple comparisons, the non-parametric Friedman test with Dunn’s post hoc correction was used. All tests were two sided. Statistical analysis was performed using Prism (GraphPad, La Jolla, CA) and OriginPro 9.1 (OriginLab).

Statistical Methods

All values are expressed as mean ± SEM unless otherwise stated. For each experimental group, sample size was chosen based on previous studies ( Carrillo-Reid et al., 2016 , Packer et al., 2015 , Rickgauer et al., 2014 ). No statistical methods were used to predetermine sample size. All recordings with no technical issues were included in the analysis. For N ≥ 10, a Kolmogorov-Smirnov normality test was used to test for normality. For N < 10, a Saphiro-Wilk normality test was adopted. In case of normal distribution, Student’s t test was used to calculate statistical significance when comparing two populations of data. For non-normal distributions, the non-parametric Mann-Whitney test or Wilcoxon signed-rank test (for unpaired or paired comparison, respectively) was used unless otherwise stated. When multiple (>2) populations of data were compared, one-way ANOVA with Bonferroni or Tukey’s honestly significant difference (HSD) post hoc test was used in case of Gaussian distribution. For non-normal distribution and multiple comparisons, the non-parametric Friedman test with Dunn’s post hoc correction was used. All tests were two sided. Statistical analysis was performed using Prism (GraphPad, La Jolla, CA) and OriginPro 9.1 (OriginLab).

Supplemental Information Document S1. Supplemental Experimental Procedures, Figures S1–S7, and Tables S1–S3 Document S2. Article plus Supplemental Information