Fluorescence imaging of large-scale neural ensemble dynamics

Abstract

Recent progress in fluorescence imaging allows neuroscientists to observe the dynamics of thousands of individual neurons, identified genetically or by their connectivity, across multiple brain areas and for extended durations in awake behaving mammals. We discuss advances in fluorescent indicators of neural activity, viral and genetic methods to express these indicators, chronic animal preparations for long-term imaging studies, and microscopes to monitor and manipulate the activity of large neural ensembles. Ca²⁺ imaging studies of neural activity can track brain area interactions and distributed information processing at cellular resolution. Across smaller spatial scales, high-speed voltage imaging reveals the distinctive spiking patterns and coding properties of targeted neuron types. Collectively, these innovations will propel studies of brain function and dovetail with ongoing neuroscience initiatives to identify new neuron types and develop widely applicable, non-human primate models. The optical toolkit's growing sophistication also suggests that "brain observatory" facilities would be useful open resources for future brain-imaging studies.

Figure 1:. Viral and transgenic techniques for labeling neurons across the mammalian brain.

(A) The TIGRE2.0 mouse transgenic strategy can achieve bright expression of a fluorescence label in a chosen cell class via the use of a genetic reporter construct that expresses both the fluorophore and a trans-activator to amplify expression. The diagram illustrates this for a double transgenic that expresses the Ca²⁺-indicator, GCaMP6f, in a Cre-recombinase-dependent manner using the Ai148 reporter mouse line from the Allen Institute for Brain Science. Cre-recombinase is expressed via a selected genetic promoter (top) and then excises two different lox-stop-lox sequences in the reporter construct (bottom). After excision, the tetracycline-regulated trans-activator (tTA2) binds to the Tet responsive element (pTRE2) and drives amplified transcription of GCaMP6f. (B) Example fluorescence image showing dopamine neurons of the substantia nigra pars compacta (SNc) from a double transgenic mouse that is a cross of the DAT-Cre driver line (DAT: dopamine transporter) and the TIGRE2.0 GCaMP6f reporter mouse line (Ai148, shown in A). Fluorescence immunostaining (anti-TH; red) confirms selective expression of GCaMP6f (green) within dopaminergic neurons. Scale bar: 100 μm. TH: Tyrosine hydroxylase. (C) By crossing more than two transgenic mouse lines, one can express both a fluorophore and an optogenetic actuator. To illustrate, the image shows a neocortical slice from a triple transgenic mouse that is a cross of the Cux2-CreER^T2 driver line, which targets layer 2/3 cortical pyramidal neurons, and two different reporter mouse lines that conditionally express GCaMP6f (green; Ai93 line) and the red-shifted opsin ChrimsonR (red; Ai167 line). Only a few cells express both constructs (yellow cells). Scale bar: 100 μm. (D) Left, A recently developed form of adeno-associated virus, AAV-PHP.eB, crosses the blood-brain barrier in many rodent strains and thus enables genetic targeting of neurons across the brain following intravascular virus injection. Diagram depicts injection of AAV-PHP.eB into the tail vein. Right, Fluorescence immunostaining reveals brain-wide expression patterns of the Ca²⁺-indicator, GCaMP6f, in a coronal section of the rat brain containing neocortex, hippocampus and thalamus. Colored boxes atop the brain section enclose areas that are magnified at right. Scale bars: 1 mm and 100 μm in the low and high-magnification images, respectively. (E) Left, Retrograde viral labeling methods allow targeting of neurons according to their axonal projection patterns. For example, using the adeno-associated virus AAV2-retro, one can label neurons with axons near the site of virus injection, irrespective of whether their cell bodies are nearby or far away. The diagram illustrates an example in which AAV2-retro is injected at a site in one coronal plane (#2; in the striatum), allowing the virus to label the cell bodies of neurons in other coronal planes that project axons to the injection site. Right, Example images taken at 4 different coronal planes (numbered as at left) revealing expression of the red fluorescent marker, tdTomato, in the cortex, amygdala and thalamus after AAV2-retro injection in the striatum. Scale bars: 800 μm (panels 1, 2) and 200 μm (panels 3, 4) (F) Top, Viral and transgenic methods can be combined to enhance targeting of specific neural populations. For instance, in a Cre-driver mouse line that targets layer 5 cortical pyramidal neurons (Rbp4-Cre), injection into the pons of a AAV2-retro virus that allows Cre-dependent expression of tdTomato (red) selectively labels layer 5 neurons in the cortex (Ctx) that project axons to the pons. AAV1-EGFP virus (green) that is co-injected into the pons labels pontine axonal output to the cerebellum (Cbl). Bottom, Fluorescence image illustrating this hybrid approach, showing cortico-pontine axons in red and ponto-cerebellar axons in green. Scale bar: 1 mm. A–C are adapted from (Daigle et al., 2018). D is adapted from (Challis et al., 2019). Fluorescence images are courtesy of M. Fabiszak/W. Freiwald lab. E and F are adapted from (Tervo et al., 2016).

Figure 2:. Chronic preparations for imaging neural activity in multiple brain areas.

(A) Top, To implant a cranial window, a portion of the skull is removed and permanently replaced by a glass coverslip (typically 3–7 mm in diameter). Bottom, Large windows provide access to multiple cortical areas, as illustrated here with a bright-field image of the brain taken through a window atop the primary visual (V1) and higher-order visual cortical areas (AM, PM, RL, AL, LM, LI). Scale bar: 1 mm. (B) Top, To image deep brain regions, gradient refractive index (GRIN) microendoscopes can be inserted to the area of interest, as illustrated here for the basolateral amygdala (BLA). Teal shading beneath the microendoscope denotes fluorescence labeling. Microendoscopes typically have diameters of 0.5–2.0 mm and lengths of 2–10 mm, and project a real image of the specimen plane to just above the microendoscope’s external face. Microendoscopes are compatible with a variety of imaging modalities, including the miniature integrated fluorescence microscope depicted in the figure. Bottom, A bright-field image of the BLA and endopiriform nucleus (EPN) taken through an implanted microendoscope. Scale bar: 200 μm. (C) In the ‘Crystal Skull’ preparation, the entire dorsal portion of the mouse cranium is permanently replaced with a curved glass window with a radius of curvature that matches that of the cortical surface. The fluorescence image shown here is a maximum projection of a 20-s-recording of neural Ca²⁺ activity acquired on a one-photon epifluorescence macroscope in a transgenic mouse (Rasgrf2-2A-dCre/CaMKIIa-tTA/Ai93) expressing GCaMP6f in layer 2/3 cortical pyramidal cells. Individual cells are visible as discrete puncta in this low magnification image. Atop the left cerebral hemisphere is a map of >30 cortical areas as demarcated in the Allen Brain Atlas (mouse.brain-map.org/static/brainexplorer). Scale bar: 1 mm (D) For simultaneous access to two or more distally separated brain areas, multiple optical ports can be created on the cranium. For example, to enable simultaneous two-photon Ca²⁺ imaging of neural activity in premotor cortex and the contralateral cerebellum, (Wagner et al., 2019) installed a pair of 3-mm-diameter windows, one above each of these two brain areas. To allow concurrent optogenetic manipulations of the disynaptic cortico-cerebellar pathway, a pair of multimode fiber implants were also implanted bilaterally targeting the pontine nuclei. Fig. 1F shows the cortico-ponto-cerebellar circuit. Scale bar: 3 mm. (E) A pair of windows, installed similarly to D, allowed concurrent one-photon fluorescence Ca²⁺ imaging of motor cortical and cerebellar neural activity in freely moving mice using a pair of miniaturized microscopes. Scale bar: 5 mm. (F) Dual-site Ca²⁺ imaging in two brain areas in the same cerebral hemisphere of a freely moving mouse. Top, one GRIN microendoscope targets the prelimbic cortex (PrL) and another microendoscope targets the CA1 subfield of the ipsilateral hippocampus. To allow the joint use of two mini-microscopes, the hippocampal microendoscope has a right-angle microprism atop its external face, allowing the two microscopes to be oriented orthogonally to avoid collision. Scale bar: 5 mm. Bottom, A pair of maximum intensity projection images of Ca²⁺ movies taken simultaneously in the two brain areas in a transgenic mouse expressing GCaMP6f (Rasgrf2-2A-dCre/CaMKIIa-tTA/Ai93). Scale bars: 100 μm. A is adapted from (Holtmaat et al., 2009) and (Stirman et al., 2016). B is adapted from (Grewe et al., 2017). C is from (Kim et al., 2016). D is from (Wagner et al., 2019). E is from (de Groot et al., 2020). F was provided by Tony H. Kim and Yanping Zhang.

Figure 3:. Two-photon mesoscopes and multi-arm microscopes for monitoring neural Ca²⁺ activity in multiple brain areas.

(A) A two-photon mesoscope made by (Sofroniew et al., 2016) has a ~5-mm-wide field-of-view (FOV). However, imaging with this system requires tradeoffs between spatial and temporal sampling. Different patterns of laser-scanning are feasible, yielding different combinations of field-of-view area, image pixel size, and frame-acquisition rate. Shown are 4 different laser-scanning patterns, in which each red trace denotes the laser beam trajectory. In patterns #1 and #2, the mesoscope acquires images across the entire accessible field-of-view, but at sub-1 Hz frame-acquisition rates or with coarse pixel spacing, as shown in the table entries below each pattern. Pattern #3 samples a conventional 600-μm-wide FOV with finer spatial resolution at a 21-Hz-frame rate. Pattern #4 sequentially samples four sub-regions at a 9.5-Hz-frame rate. In the table, d′ values are normalized to those of Pattern #3 with all other optical conditions held fixed. (B) Example images from the neocortex of a Thy1-GCaMP6f transgenic mouse, taken with the mesoscope of A. Left, An image of the brain acquired by sweeping the laser across much of the accessible FOV using scanning pattern #2 shown in A. Highlighted are 4 sub-regions in which Ca²⁺ imaging was performed at higher spatiotemporal resolution. Scale bar: 1 mm. Right, Images from each of the 4 regions highlighted at left, taken with scanning pattern #4 shown in A. Orange circles indicate regions of interest corresponding to individual neurons. Scale bar is 200 μm and applies to all 4 images. (C) A different two-photon mesoscope (Rumyantsev et al., 2020) uses a 4 × 4 array of laser illumination beams and a corresponding array of photodetectors (PMT: photomultiplier tube) to sample 16 different sub-portions of the specimen plane in parallel. Each beam sweeps across a 500-μm-wide area, enabling Ca²⁺ imaging over a 2 mm × 2 mm area with micron-scale pixel resolution at frame rates up to 20 Hz. (D) Fluorescence image of the primary visual cortex (V1) of a triple transgenic mouse (Rasgrf2-2A-dCre/CaMKIIa-tTA/Ai93) expressing GCaMP6f in layer 2/3 cortical pyramidal cells, taken using the mesoscope of C. Identified neurons are shown in green. Dotted white lines demarcate the 16 image tiles. Acquisition parameters are listed below the image. Top inset, A magnified view of the image portion outlined in blue. Scale bar: 100 μm. Bottom inset, Spatial profile of one GCaMP6f-expressing neuron, demonstrating subcellular spatial resolution. Scale bar: 100 μm. (E) Schematic of a dual-axis two-photon microscope for imaging a pair of distally separated brain areas. Each microscope ‘arm’ has 3 translational and 2 rotational mechanical degrees-of-freedom (DOF), enabling flexible positioning of 2 microscope objectives. Illumination beams are simultaneously scanned in the two FOVs using two independent sets of scanning mirrors. This system can image pairs of brain areas that are separated by as little as ~1 mm (Lecoq et al., 2014), or >9.5 mm apart (as in F). (F) Concurrently acquired pair of images of motor cortical layer 5 neurons and cerebellar granule cells, obtained using the dual-axis microscope of E and the multi-port surgical preparation of Fig. 2D. GCaMP6f was expressed in the 2 brain regions using a quadruple transgenic mouse (Rbp4-Cre/Math1-Cre/Ai93/ztTA). Scale bars: 100 μm (motor cortex) and 50 μm (cerebellum). A and B are adapted from (Sofroniew et al., 2016). C and D are adapted from (Rumyantsev et al., 2020). E is adapted from (Lecoq et al., 2014). F is adapted from (Wagner et al., 2019).

Figure 4:. Photon detection rate per cell is a key determinant of single spike sensitivity.

In two-photon microscopy, the rate at which fluorescence is captured from individual cells varies with the illumination power and the time per image frame that the laser dwells on each cell (cellular duty ratio). In a raster-scanning microscope, when a user increases the total area in tissue that is scanned with the laser at a fixed laser power, the sampling time per neuron and thus the spike sensitivity both decline. (A) To study how the ability to detect single action potentials varies with the scanning parameters of two-photon Ca²⁺ imaging, (Huang et al., 2020) performed simultaneous loose-patch electrical and optical Ca²⁺ recordings of neural activity in transgenic mice (Cux2-CreER^T2/CaMKIIa-tTA/Ai93) expressing the Ca²⁺ indicator GCaMP6f in layer 2/3 visual cortical pyramidal cells. Panels A–C compare results from two different imaging configurations, a low-magnification configuration in which a 400-μm-wide area of tissue was scanned at 30 Hz, representative of conventional two-photon Ca²⁺ imaging of neural population activity, A, and a high-magnification configuration in which a 20-μm-wide area containing only a single cell was scanned at 158 Hz, A inset. Dashed purple lines outline the glass microelectrode (B) Simultaneously acquired traces of Ca²⁺ (blue) and electrical (purple) activity from an individual neuron under the two different imaging configurations shown in A, at the same illumination power. Action potentials are visible in the electrical traces as discrete impulses. (C) Assessment of spike-sensitivity in the 2 imaging configurations of A, B. Top, Box-and-whisker plots showing the fractional change in [Ca²⁺]-related fluorescence, ΔF/F, as a function of the number of spikes that an example cell fired in a 50 ms interval. For a given number of spikes, median values of the ΔF/F response (horizontal red lines) are comparable in the 2 configurations, but variances are greater in the low-magnification case due to fewer detected photons per cell. Boxes span 25th to 75th percentiles; whiskers extend to 1.5 times the interquartile distance; red data points are outliers. Bottom, Normalized histograms showing the number of instances in which a given ΔF/F response value was observed in a 50-ms-interval with no spikes (gray data) or a single spike (blue data). The spike detection fidelity (d′) depends on the separation between the pair of histograms. The distributions are modestly different in the high-magnification configuration (bottom left), but are nearly indistinguishable in the low-magnification configuration (bottom right). (D) Signal detection theory provides predictions of the spike detection fidelity d′ under different imaging conditions. The graph shows a contour plot of d′ for GCaMP6f as a function of the two-photon imaging fieldof-view and frame-acquisition rate, with the illumination power held constant. Black lines are equicontours of d′. Points marked with ☆ symbols correspond to the two different imaging configurations of A–C. To produce this plot, we set the single-spike ΔF/F response and the fluorescence decay time constant of GCaMP6f to be 19% and 200 ms, per (Chen et al., 2013). We then scaled the d′ values to fit the true positive rate (TPR) of single spike detection (~54%) found by (Huang et al., 2020) in the high-magnification imaging configuration, given a 5% false positive rate (FPR). Signal detection theory then predicts a greatly diminished, ~6% TPR for single spike detection in the low-magnification configuration, consistent with the experimental results in C. (E) In conventional two-photon microscopy in which the laser is scanned in a raster pattern, the laser samples many points in the image without labeled neurons, lowering the cellular duty ratio. To improve the duty ratio, random access scanning selectively addresses a set of user-selected cells. Top, A schematic depiction of a form of random access scanning (‘chessboard’ scanning), in which a pair of acousto-optic deflectors scans small, discontiguous patches of tissue (red planes) that each contain an individual cell body. Bottom, Single-frame ‘chessboard’ image of GCaMP6f-expressing pyramidal cells from mouse visual cortex. The 136 neurons shown were imaged at 11.14 Hz. Each image patch is 20 μm wide. (F) An alternative means of increasing the fluorescence flux per cell in multiphoton Ca²⁺ imaging involves an adaptive excitation source (AES), a laser that allows variation of the illumination power based on the image content. By gating laser emission such that only regions-of-interest (ROIs) with cell bodies are illuminated (bottom left), one can increase the efficiency with which the illumination power generates productive fluorescence, as compared to conventional two-photon imaging of the same specimen (top left). For the 3 ROIs in the bottom left panel, the right 6 graphs show GCaMP6s fluorescence traces with (bottom row) and without (top row) the use of the AES. When using the AES, the fluorescence per frame per cell, F₀, increased about 7-fold on average, even though the net illumination power delivered to the brain was ~4.5 times lower (18 mW with AES). Frame rate was 30 Hz in both cases. Scale bars: 100 μm. A–C are adapted from (Huang et al., 2020), using datasets #103920 and #103922 from the Allen Institute for Brain Science (portal.brain-map.org/explore/circuits/oephys). E is adapted from (Szalay et al., 2016). F is adapted from (Li et al., 2020).

Figure 5:. Imaging neural activity using parallel readout strategies.

(A) Schematic of an epi-fluorescence mesoscope based on an array (5 × 7) of cameras. This system allowed 30-Hz-imaging of neural Ca²⁺ activity across the neocortical surface (10 mm × 12 mm FOV) with 0.8-μm-wide pixels. Inset, Maximum projection image of the neocortex of a Thy1-GCaMP6s mouse; the boundaries between image tiles are just barely visible. Scale bar: 1 mm. (B) Top left, A high-speed camera and an array of 400 laser focal spots that is swept laterally across the specimen plane enable high-speed (0.1–1 kHz) two-photon imaging of neural activity. Top right, This approach enabled imaging of dendritic Ca²⁺ spiking by cerebellar Purkinje cells at a 100-Hz-frame rate across a 450 μm × 300 μm field-of-view. Scale bar: 50 μm. Bottom, In comparison to Ca²⁺ imaging data taken by conventional two-photon microscopy (green data points), multi-beamlet two-photon imaging (red data points) achieved ~10-fold better Ca²⁺-spike timing estimation precision (y-axis), with comparable or better values of spike detection fidelity (x-axis). (C) The point-spread function (PSF) of a two-photon microscope describes the spatial distribution of two-photon excited fluorescence relative to the centroid of the laser focal spot. Left, In a conventional two-photon microscope, a laser beam with a cross-sectional intensity profile that is approximately Gaussian yields a PSF with sub-micron lateral width (x axis) and micron-scale axial (z axis) extent. Middle, By using a temporally focused beam for two-photon excitation, the PSF can be enlarged laterally while retaining micron-scale axial sectioning. Right, By using Bessel-beam illumination for two-photon excitation, the PSF can be axially elongated to the hundred-micron-scale while retaining sub-micron widths. (D) Two-photon imaging of GCaMP6s activity in neural dendrites and spines in mouse visual cortex using Bessel-beam illumination. Left, Mean projection image of a volumetric image stack spanning 60 μm in depth, in which each image slice was acquired sequentially using a Gaussian beam and a PSF with a 1.4 μm axial FWHM. Colors encode axial depths below the pial surface. Right, Image of the same dendrites and spines obtained by scanning a Bessel beam that yielded a PSF with a 35 μm axial FWHM. Imaging with the Bessel-beam led to ~20-fold higher temporal resolution than the conventional approach. Scale bar: 20 μm. (E) One can track neural Ca²⁺ activity in multiple axial planes concurrently by first mapping neurons’ locations and then using this information to unmix neural Ca²⁺ activity traces from superposed multi-plane Ca²⁺ data. Panel 1, First, conventional two-photon microscopy is used to map the neurons’ locations (3 planes denoted in different colors). Panel 2, Holographic laser illumination provides multiple focal spots, one in each of the 3 axial planes. The 3 spots are laterally scanned in parallel, allowing the 3 planes to be simultaneously sampled. This yields superposed images of fluorescence from the different planes. Panel 3, Ca²⁺ activity traces of individual cells are recovered from the superposed data using their previously determined shapes and locations. Overall, this strategy increases the volumetric imaging rate by a factor of N, where N is the number of planes sampled in parallel. (F) Anatomic data and statistical estimation can also be used to boost the speed at which neural dynamics are tracked in one axial plane. Panel 1, Using conventional two-photon microscopy, one acquires a high-resolution image of a specimen that is sparsely labeled with a neural activity indicator. Structures of interest, such as dendrites, are divided into discrete segments, each of which will be assigned an individual activity trace. Panel 2, To acquire activity data, 4 lines of laser illumination with different orientations are swept sequentially across the specimen plane of interest. In this way, multiple neural segments are sampled in parallel. Panel 3, Using a statistical estimator that accounts for the segments’ morphology and indicator kinetics, activity traces are reconstructed for each segment. This approach allowed reconstructions of glutamate release dynamics in neocortical dendrites across a 250-μm-wide FOV. A is adapted from (Fan et al., 2019). B is adapted from (Zhang et al., 2019). D is adapted from (Lu et al., 2017). E is adapted from (Yang et al., 2016). F is adapted from (Kazemipour et al., 2019).

Figure 6:. Integrated two-photon optogenetics and two-photon Ca²⁺ imaging.

(A) Diagram of an instrument that combines conventional two-photon microscopy with basic two-photon optogenetics (OG) capabilities. Separate lasers provide illumination for imaging and optogenetic control, and there are two different pairs of laser scanners for steering each focal spot. In A, C and G the dashed boxes enclose components comprising a standard two-photon microscope. Key components needed for optogenetic studies are shown outside the boxes. (B) Using the system of A, (Jennings et al., 2019) stimulated up to 20 neurons selected based on their Ca²⁺ activity patterns. Left, A two-photon image of mouse orbitofrontal cortical (OFC) neurons expressing the Ca²⁺ indicator, GCaMP6m, and a red-shifted excitatory opsin, bReaChES. Red outlines mark cells chosen for optogenetic stimulation; green outlines mark nearby cells that were not targeted. Scale bar: 100 μm. Middle, A schematic of the boxed region shown at left, with cells depicted as colored circles. The scanner controlling the optogenetics beam sequentially targeted each of the numbered cells and applied a 1-ms-duration spiral scan of the focal spot to each cell body. During the ~0.12 ms time needed for the scanners to switch between cells, the optogenetic illumination was electro-optically shuttered. Right, Ca²⁺ activity traces of targeted (red) and non-targeted (green) cells. Red shading denotes intervals when optogenetic stimulation was applied. (C) Schematic of a two-photon imaging system that can optogenetically manipulate multiple cells in parallel by applying spiral laser-scans to each of them concurrently. In addition to the apparatus of A, the system includes a programmable spatial light modulator (SLM) that splits the optogenetics laser beam into multiple beamlets, each of which targets one user-selected cell. The zero-order block blocks undiffracted light from the SLM. (D) Using the system of C, (Packer et al., 2015) stimulated 10–20 neurons at once. Cell-attached electrical recordings revealed the temporal precision with which two-photon optogenetics evoked spiking. Left, A two-photon image of somatosensory cortex in which layer 2/3 pyramidal cells expressed GCaMP6s and the red-shifted excitatory opsin C1V1. Red spirals mark cells that were optogenetically targeted with parallel spiral scanning. A glass microelectrode was used to perform a loose patch electrical recording of one of the targeted neurons. Scale bar: 100 μm. Top right, A set of electrical traces of neural activity from 10 trials in which the cell was optogenetically stimulated using spiral scanning (red shaded interval). Bottom right, The electrical traces permit quantification of the mean latency to spike and the timing jitter across trials, for different durations of spiral scanning and either 10 or 20 targeted cells. (E) Using a soma-targeted variant of the excitatory opsin ChRmine and a larger version of the instrument in C capable of activating more cells, (Marshel et al., 2019) stimulated >100 user-selected neurons by rapidly switching between different sets of 20–30 cells. Left, Timing protocol for targeting 6 different sets of neurons. Each set of cells was stimulated with a 0.21-ms-duration spiral scan, and a different set was targeted every millisecond. Right, z-scored Ca²⁺ activity traces for the 6 different sets of cells, with each line showing an optogenetically evoked response of an individual cell that was successfully activated. The number of cells that were successfully activated out of the total targeted is shown for each set. In total, 124 cells out of 160 cells targeted were successfully activated. (F) Schematic of a two-photon microscope in which holography and temporal focusing allow two-photon optogenetic control of multiple cells that are each illuminated with a disk of laser light. Note the lack of a scanner in the optogenetics illumination pathway. Using this system, (Mardinly et al., 2018) optogenetically manipulated up to ~50 neurons at once. (G) Electrophysiological validation of the system of F. Left, Using a loose-patch electrical recording (glass microelectrode filled with green dye) of a parvalbumin-positive (PV) interneuron expressing the fast excitatory opsin Chronos and the red fluor mRuby2, (Mardinly et al., 2018) measured the reliability with which they could optogenetically evoke single spikes. Middle, Raster plots of activity from an individual PV neuron that was stimulated at time points separated by Poisson-distributed intervals (mean rate of stimulation: 30 Hz). Green dots mark instances in which stimulation evoked an action potential. Orange dots mark failures to spike. Grey dots mark spontaneously emitted spikes. Right, Probability of successful spiking per stimulation attempt in PV, somatostatin (SOM) and vasoactive-peptide (VIP) interneurons, as a function of the stimulation rate. A and B are adapted from (Jennings et al., 2019). C and D are adapted from (Packer et al., 2015). E is adapted from (Marshel et al., 2019). F and G are adapted from (Mardinly et al., 2018). Panels C and F were redrawn using graphical elements from (Jennings et al., 2019).

Figure 7:. In vivo voltage imaging at single spike resolution.

(A) Simultaneous in vivo one-photon optogenetic (OG) stimulation and one-photon fluorescence voltage imaging. Top, Fluorescence image of 3 interneurons in mouse neocortical layer 1 that co-express the near-infrared voltage-indicator SomArchon and the blue light-activated excitatory opsin SomCheRiff from a bicistronic AAV construct. Scale bar: 20 μm. Bottom, Simultaneously acquired fluorescence traces from the 3 cells. Blue shading marks the period when optogenetic stimulation was applied. Images were taken at 1 kHz frame rate. (B) Top, In the mushroom body of adult Drosophila melanogaster, the PPL1-α’2α2 and PPL1–2α’1 neurons expressed the red-fluorescent FRET-opsin voltage indicator, VARNAM. The MBON-α2sc neuron expressed the green-fluorescent FRET-opsin, Ace2N-mNeon. Bottom, Owing to the anatomic separation of the PPL1-α’2α2 and PPL1–2α’1 cells and the different emission wavelengths of the two indicators, optical voltage traces of spiking activity could be acquired concurrently from all 3 individual neurons. Images were acquired at 500 Hz. (C) Epi-fluorescence voltage imaging of layer 1 neocortical interneurons in a NDNF-Cre driver mouse line using the soma-targeted (ST) version of the chemical genetic voltage-indicator Voltron₅₂₅. Top left, Fluorescence image of Voltron₅₂₅-ST-expressing layer 1 interneurons in the visual cortex. Cortical expression of Voltron clearly exceeds the 1064 μm × 266 μm FOV (boxed area) allowed by the camera when operating at 400 Hz. Scale bar: 1 mm. Top right, Magnified view of the area boxed at left. Scale bar: 100 μm. Numbers indicate cells whose voltage activity was imaged simultaneously at a 400 Hz frame rate. Bottom left, Optical voltage traces for 10 cells from a portion of a 3-min-recording. Bottom right: Magnified view of the traces for cells #4–6, for the time period enclosed in the purple box at left. Black dots mark individual action potentials. (D) Two-photon voltage imaging in the mouse visual cortex using a high-speed raster scanning. Top, 50 μm × 250 μm area of brain tissue showing 4 cells, 75 μm beneath the pial surface, that express a soma-targeted voltage indicator, ASAP3-Kv, under the control of the synapsin promoter. Scale bar: 10 μm. Bottom, Optical voltage traces of spontaneous activity from the 4 cells, including a period of burst firing in one cell (highlighted in purple), taken at 1 kHz frame rate. (E) Two-photon voltage imaging of 3 pyramidal cells in mouse neocortical layer 5 that express ASAP3-Kv, performed using random-access laser-scanning. Top left, A set of image slices acquired at different axial (z) depths in tissue. Boxed areas indicate apical dendrites, and the color-corresponding arrows mark cell bodies of the same neurons. Top right, A single image slice, showing apical dendrites of 3 individual neurons (colored boxes) at a depth of z = 421 μm. Bottom, Traces show the voltage activity of the 3 color-corresponding neurons shown above, sampled at their apical dendrites (3.33 kHZ per cell). Red ticks mark occurrences of dendritic spikes. For cell #3, the horizontal grey lines denote voltage levels corresponding to up- and down-voltage states. A is adapted from (Fan et al., 2020). B is adapted from (Kannan et al., 2018). C is adapted from (Abdelfattah et al., 2019). D is adapted from (Wu et al., 2020). E is adapted from (Villette et al., 2019).