Decision letter: Reconstruction of genetically identified neurons imaged by serial-section electron microscopy

Field: Neuroscience (Life Sciences)

Article Figures and data Abstract eLife digest Introduction Results and discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Resolving patterns of synaptic connectivity in neural circuits currently requires serial section electron microscopy. However, complete circuit reconstruction is prohibitively slow and may not be necessary for many purposes such as comparing neuronal structure and connectivity among multiple animals. Here, we present an alternative strategy, targeted reconstruction of specific neuronal types. We used viral vectors to deliver peroxidase derivatives, which catalyze production of an electron-dense tracer, to genetically identify neurons, and developed a protocol that enhances the electron-density of the labeled cells while retaining the quality of the ultrastructure. The high contrast of the marked neurons enabled two innovations that speed data acquisition: targeted high-resolution reimaging of regions selected from rapidly-acquired lower resolution reconstruction, and an unsupervised segmentation algorithm. This pipeline reduces imaging and reconstruction times by two orders of magnitude, facilitating directed inquiry of circuit motifs. https://doi.org/10.7554/eLife.15015.001 eLife digest Neurons connect with each other to form complex circuits that underlie mental activities. Mapping these connections to obtain a so-called wiring diagram is an essential step in learning how the brain works. The only way to do this precisely enough is by using electron microscopy. However, this technique is so time-consuming that thousands of hours of work are typically required to image even the smallest of tissue samples. Electron microscopes fire beams of electrons at tissue samples, and detect the scattering of the electrons. Stains are used to make specific neurons less permeable to electrons, or more “electron dense”. Labeled cells scatter more electrons, which increases the contrast of the images. In an approach called serial-section electron microscopy, a tissue sample is first cut into extremely thin sections. These are imaged individually, and the images are then pieced together to reconstruct the sample. Joesch et al. have now developed a new procedure – named ARTEMIS – that uses a combination of multiple techniques to speed up the mapping of neurons and their connections. ARTEMIS makes use of genetic engineering, serial-scanning electron microscopy, an enhanced chemical staining procedure and a new image processing approach. First, gene technology is used to selectively stain specific types of neurons in mice and flies. Then, a tissue sample is collected and treated with a chemical that enhances the electron density of the stained neurons, without disrupting the tissue’s structure. Next, a labeled target neuron is imaged at relatively low resolution to reveal its overall structure. Small areas of that neuron are then re-imaged at higher resolution to map the connections between neurons. Lastly, an algorithm pieces together the individual images to produce a reconstruction of the cell. This pipeline of steps reduces the time required to map the shapes and connectivity of neurons with electron microscopy by some two orders of magnitude. This should enable neuroscientists to obtain more rapid insights into the roles of specific neural circuits in the brains of healthy animals, and to identify cases where this wiring goes awry and leads to disease. https://doi.org/10.7554/eLife.15015.002 Introduction Relating neural circuits to the computations they perform requires mapping patterns of structural and functional connectivity among neurons. Innovative light microscopic methods such as GRASP, trans-synaptic viral tracing and super-resolution imaging enable visualization of synapses made on or by identified neurons (Wickersham and Feinberg, 2012; Tønnesen and Nägerl, 2013). At present, however, only electron microscopy (EM) provides sufficient resolution to visualize the complete complement of synapses that neurons form and receive. Indeed, large-scale reconstructions from serial sections have provided deep insights into neuronal circuit principles (Briggman et al., 2011; Bock et al., 2011; Takemura et al., 2013; Morgan et al., 2016) . The optimal strategy is to collect serial sections containing all circuit elements and image them at nanometer resolution. Processes in the imaged volumes are then segmented to reconstruct the neurons (or parts of neurons) they contain. Advances in sectioning, imaging and segmentation methods make so-called 'saturated' reconstruction of volumes around 1000 μm3 feasible (Kasthuri et al., 2015). Even these modest volumes remain challenging, however, and when multiple samples must be compared -e.g., controls vs. mutant or treated vs. untreated animals - this approach is currently out of reach (Plaza et al., 2014). An attractive alternative is 'sparse' reconstruction of specific cells within a fully imaged volume. For example, neuronal activity can be monitored using calcium indicators, then neurons with particular patterns of activity can be relocated in thin sections and reconstructed (Briggman et al., 2011; Bock et al., 2011). This method is, however, technically demanding and infeasible in many tissues. We therefore devised an alternative approach to sparse reconstruction that relies on marking specific cells with an electron-dense tracer. We then exploit the high contrast provided by the tracer to speed up imaging and reconstruction, which are currently the rate-limiting steps in connectomic analysis. Our pipeline includes the following series of steps: (a) tagging a specific cell type with a genetically encoded EM tracer, (b) enhancing the electron-density of the stain without compromising ultrastructure of the surrounding tissue, (c) imaging the cell rapidly at relatively low resolution, (d) re-imaging selected small volumes at higher resolution to map connectivity and (e) segmenting the cell using a novel algorithm that is reliable, fast and does not require computationally intense pre-training. Together, the gains from targeted reimaging and unsupervised segmentation decrease the time required for reconstruction by over two orders of magnitude. We call the method ARTEMIS for Assisted Reconstruction Technique for Electron Microscopic Interrogation of Structure. Results and discussion A classical ultrastructural tracer is horseradish peroxidase (HRP), which catalyzes the formation of a 3,3’-diaminobenzidine (DAB) polymer; the polymer binds osmium and is thereby rendered electron-dense. Recombinant HRP is enzymatically inactive in the cytosol because it fails to form disulfide bonds or become glycosylated, but this limitation can be overcome by directing the protein to topologically extracellular compartments such as vesicles (Li et al., 2010; Atasoy et al., 2014; Schikorski et al., 2007). We therefore generated an HRP variant that was codon-optimized, mutated to increase activity, and fused to an endoplasmic reticulum targeting sequence (erHRP). We also tested derivatives of plant ascorbate peroxidases called APX and APEX2, which are active in the cytosol (Martell et al., 2012; Lam et al., 2015). Initial studies using cultured HEK293 cells confirmed that all three constructs generated active peroxidase in the transfected cells (Figure 1a,b and data not shown). Figure 1 with 5 supplements see all Download asset Open asset Enhanced staining of genetically encoded tags for EM. (a–b) Bright-field images of HEK-cells rendered photon-dense by DAB polymerization, catalyzed either by APEX2 tagged with a nuclear export signal (APEX2NES) (a) or endoplasmic reticulum tagged HRP (erHRP) (b). (c–d) Bright-field images of direction-selective retinal ganglion cells (ooDSGC) expressing APEX2NES (c) or erHRP (d) and rendered photon-dense as in (a). (e) Enhanced staining strategy. (f–g) EM-micrographs of HEK-cells rendered electron-dense after DAB-polymerization as in (a). A standard EM staining protocol showed no detectable cytosolic DAB-polymer staining (f), whereas the addition of a reduction step dramatically enhanced DAB-polymer staining (g). (h) Ribbon synapses in the outer plexiform layer; ultrastructure is well preserved after reduction. (i) EM-micrograph of an ooDSGC soma (arrowhead) rendered electron dense after tissue reduction next to a non-expressing RGC (asterisk). (j) Close-up of cytosolic APEX2 staining. (k) Dendritic processes expressing APEX2 (asterisk) contacted by a presynaptic partner (arrowhead) in the inner plexiform layer. (l) Axonal long-range projections of an APEX2-expressing ooDSGC in the superior colliculus (asterisk) with a postsynaptic target (arrowhead). (m) erHRP-expressing (arrowhead) RGC next to a non-expressing RGC (asterisk). (n,o) Close-up of erHRP staining of a J-RGC soma (n) and dendrite (o) (arrowhead point to presynaptic partners). (c,d,i-l) are from Cart-cre mice; (m–o) from Jam-B-creER mice. Scale bars: (a–d) :25 μm; (f–g): 10 μm; I, m : 5 μm; h, j−l, n,o : 500 nm; https://doi.org/10.7554/eLife.15015.003 For selective expression in molecularly-identified cells, we generated adeno-associated viral (AAV) vectors in which expression of erHRP, APX or APEX2 required Cre-dependent recombination. These were used to infect retinas of transgenic mice in which specific retinal ganglion cell (RGC) types expressed Cre recombinase (ooDSGCs in Cart-cre [Kay et al., 2011]) or tamoxifen-activated Cre (J-RGCs in JAM-B-creER [Kim et al., 2008; Joesch and Meister, 2016]). Two to four weeks after infection, retinas were reacted with DAB and H2O2 and examined light-microscopically, revealing intense labeling of RGCs (Figure 1c,d). Retinas were then processed for EM (see Methods). Unexpectedly, levels of electron-dense precipitate were so low that stained processes could not be traced reliably (Figure 1—figure supplement 1a,b). Numerous alterations to balance peroxidase activity and ultrastructural quality failed to improve matters: when ultrastructure was adequately preserved, staining for peroxidase was poor, and when reaction product was adequate, synaptic structures were poorly preserved. This difficulty may have been less apparent in previous studies using injected native HRP, which has substantially higher activity than the recombinant proteins we use. The reason for the difference between light and electron microscopic results is likely that the opacity and electron density of the DAB polymer arise in different ways: its polycyclic structure renders it photon absorbent, but its electron-density results from redox reactions with osmium (Bahr, 1954). We reasoned that radicals produced during the long peroxidase reaction might oxidize the relevant functional groups in the polymer, leaving it photon-absorbent but inert to osmium. If this were true, reduction of functional groups on the polymer could restore reactivity to osmium (Figure 1e). We tested this hypothesis in transfected HEK cells. When cells were treated with the protocol we had used for retina, the precipitate was clearly visible by light but not electron microscopy. However, when the HEK cells were treated with a mild reducing agent (5 mM sodium hydrosulfite) between the peroxidase reaction and osmication, they were highly electron-dense (Figure 1f,g). This was the case using either conventional osmium staining or an enhanced 'double osmium' staining protocol (rOTO), although the latter showed a slight improvement probably due to the change in the redox state of osmium tetroxide (Figure 1—figure supplement 1c–f; see Methods). A similar effect was observed in erythrocytes, in which endogenous heme catalyzes the DAB reaction (Figure 1—figure supplement 1g–j). When the reduction protocol was applied to retinas, we were able to visualize RGCs that had been tagged with APX, APEX2NES (APEX2 fused to a nuclear export signal) or erHRP (Figure 1i–o, Figure 1—figure supplement 2 and 3). As expected, APX and APEX2NES labeled the cytoplasm diffusely (Figure 1j) while erHRP labeled membrane-bound intracellular compartments (Figure 1n). Thus they could be used as orthogonal labels, although we have not yet pursued this application. The strength of the signal allowed us to identify small stained dendritic profiles (Figure 1k,o) and to view the terminals of RGCs in the superior colliculus, approximately 1 cm from the somata (Figure 1l). Remarkably, the reduction protocol actually improved the visualization of the ultrastructure irrespective of peroxidase expression or DAB treatment (Figure 1h). Part of the improvement resulted from an increase in the reactivity of membranes to osmium, thereby enhancing membrane-cytoplasm contrast (Figure 1—figure supplement 4). This improvement is also visible in the staining strength of synaptic densities (Figure 1—figure supplement 4a,b). When we asked blind-to-condition observers to judge the quality of synapses between both conditions, the reduced tissue was selected ~3 times more frequently than unreduced tissue (Figure 1—figure supplement 4e). Thus, rather than sacrificing ultrastructure for reactivity or vice versa, this protocol improved both. For the approach to be useful, it is essential that peroxidase expression does not affect synapse formation and that synaptic partners of peroxidase expressing cells can be identified. When analyzing our datasets, we could not detect any structural differences between tissues expressing either of the peroxidases. The number of synapses counted in peroxidase expressing and control sections was also similar, 0.32 and 0.34 synapses / μm2, respectively. To test whether the electron dense precipitate hinders reliable synaptic identification, we characterized the synaptic connections received and made by APEX2 or erHRP expressing retinal ganglion cells (Figure 1k,o; Figure 1— figure supplement 5). Cells presynaptic to APEX2 or erHRP could be clearly identified based on ultrastructural details (Figure 1—figure supplement 5a,c). Postsynaptic partners of APEX2-expressing cells could also be identified. Moreover, synaptic vesicles of APEX2-expressing neurons could be detected in presynaptic terminals because they were unstained, and thereby contrasted with the electron-dense cytosol. However, the endoplasmic reticulum did not regularly extend to axonal terminals, making it difficult to identify postsynaptic partners of erHRP expressing ganglion cells. To test the generality of the method we expressed APEX fused to GFP in direction-selective tangential cells of Drosophila melanogaster (Joesch et al., 2008) (DB331-Gal4 → UAS-APEX-GFP; see Methods). This driver line expresses mainly in 6 vertically sensitive and 3 horizontally sensitive tangential cells. This expression pattern was apparent using either GFP fluorescence or the polymerized DAB to mark the cells (Figure 2a,b). We were also able to visualize the electron dense precipitate of these processes in the fly’s optic lobes (Figure 2c,d). Although our method was optimized for mammalian tissue, ultrastructural detail was reasonable (Figure 2e) and allowed the identification of synaptic contacts made by and onto APEX expressing cells (Figure 2f–h). Figure 2 Download asset Open asset APEX expressing interneurons in Drosophila melanogaster. (a) Fluorescent image of a brain of Drosophila melanogaster (DB331-Gal4 → UAS-APEX-GFP) expressing APEX in direction-selective lobula plate tangential cells (LPTCs). (b) Bright field image of a similar plane from another (DB331-Gal4 → UAS-APEX-GFP) fly, labeled with DAB. (c) Electron micrograph of a frontal brain section (30 nm thickness) containing electron dense staining in axonal processes of LPTCs (arrowhead and framed box). (d) Enlarged view of the framed region in (c). Arrows point to electron dense processes. (e) Unstained synaptic terminal, showing quality of ultrastructural detail. (f) Synaptic nerve terminal in an APEX-positive process, identifiable by contrast-reversed vesicles. (g,h) APEX-positive postsynaptic processes, identifiable by the presence of adjacent vesicle-laden, T-bar-containing terminals. Scale bars: a–c :100 μm; d :10 μm; e–g : 500 nm. https://doi.org/10.7554/eLife.15015.009 To implement sparse reconstruction, we used the ATUM (Hayworth et al., 2014) (automated tape-collecting ultra-microtome) to serially section retinas containing either APEX2NES-expressing retinal ganglion cells (J-RGCs) or retinal interneurons (starburst amacrine cells (SACs)) labeled using Choline Acetyltransferase-Cre (Rossi et al., 2011). Because we could identify small peroxidase-stained neurites at overview resolution (20–30 nm per pixel), we tested the minimal requirements to reconstruct both cell types at these resolutions. Rapidly imaging every 10th section (270 nm separation), at 30 nm resolution was sufficient for mapping a J-RGC dendrite (144 sections covering > 1 × 107 µm3, imaged in 22 hr; Figure 3a,b). By comparison, imaging the same volume at high resolution (4 nm/pixel) would have taken ~2500 hr on the same microscope. Manual reconstruction of the J-RGC was straightforward and took 2 worker-hours (Figure 3c). To extend this result to another cell type, we imaged all 1260 sections from a bloc with APEX2NES-positive SACs and reconstructed SAC processes in this volume (Figure 3d–f). Thus, we can reliably find and reconstruct genetically identified cells. Figure 3 Download asset Open asset Targeted interrogation of serial-section EM volumes in mouse retina. (a). Overview-resolution EM volume containing J-RGCs expressing APEX2NES. Every 10th section was imaged at 30 nm/pixel. (b) Two consecutive sections containing APEX2NES expressing J-RGC processes (arrows). (c) Reconstructed J-RGC. Note the reduced complexity of the dendritic field due to the subsampling of the overview dataset. (d) Overview-resolution EM volume that contains SACs expressing APEX2NES. The sections were imaged at 20 nm/pixel. (e) Two consecutive sections showing a SAC-SAC contact (arrowheads). (f) Reconstruction of the SAC-SAC interaction in (e); arrowhead indicates contact shown in e. (g–h). Comparison of ultrastructural detail visible at 20 nm/pix (g) or 4 nm/pix (h). Although both images contain ribbon synapses, the low-resolution image (g) lacks the required resolution to identify them. Arrowheads point to ribbons. (i) High-resolution (4 nm/pixel) EM subvolume selected from (d). (j) Two high-resolution EM-micrographs containing SAC-processes expressing APEX2NES receiving synaptic contacts (arrowheads). (k) Reconstruction of the SAC plexus with its characteristic dendritic fasciculation and net-like structure. Asterisk indicates contact and arrowheads the synaptic inputs shown in j (top panel). Scale bars: b: 5 μm; c: 50 μm; e, j: 1 μm; g–h: 200 nm. https://doi.org/10.7554/eLife.15015.010 Although 20 nm resolution is sufficient for viewing neurites, it does not allow optimal visualization of synaptic contacts (Figure 3g–h). However, sections generated using ATUM are collected on wafers, and can be re-imaged; a program, Wafer-Mapper (Hayworth et al., 2014), facilitates returning to the same place on a given section with micron-level precision. This feature allows targeted imaging at high resolution of areas chosen from the rapidly acquired, lower resolution reconstruction, thereby substantially reducing imaging time. To test this multi-scale approach, we focused on the SACs, whose dendrites can be less than 100 nm in diameter. We selected a heavily-labeled ~5 × 105 µm3 volume from the overview-resolution reconstruction of ~2 × 107 µm3 and acquired a high-resolution (4 nm/pixel) data set in 250 hr (Figure 3i). Compared to a completely high-resolution approach, this amounts to a 40-fold reduction in imaging time. More importantly, it expedited reconstruction, the current time-limiting step in connectomics (Plaza et al., 2014), by constraining efforts to defined regions of interest. We manually traced and reconstructed the SAC plexus in this volume, observing multiple SAC-SAC interactions, dendritic fasciculation in the SAC plexus and dendritic branching (Figure 3j,k and data not shown). Finally, to further expedite the reconstruction pipeline, we developed an algorithm that detects and segments APEX2-positive processes, taking advantage of the high contrast rendered by the DAB polymer. Our algorithm is fast, parameterized and does not rely on the supervised machine learning training currently required for lower contrast (Kasthuri et al., (Figure Materials and We tested the algorithm using sections from the high-resolution (Figure 3i). We a set of APEX2 and then compared these results to We a of for the segmentation of the algorithm (Figure segments were small × and were necessary to with the of in can make connections in regions with small To test the of the algorithm for processes, we segmented and reconstructed a dendritic of a amacrine cell. Our algorithm could reconstruct the of the dendritic processes and of the manually segmented details (Figure The advantage of this approach is processing two orders of than based on neural for (Kasthuri et al., et al., 2015). in the neural require at thousands of for each new in our algorithm unsupervised with a small set of (see and the high of the cells is the for the of this which that it could be used for reconstruction of structures in any Thus, although synaptic may be by the electron-dense our methods are for rapid reconstruction of targeted neuron and Figure 4 Download asset Open asset segmentation and reconstruction algorithm for APEX2 processes. (a) The algorithm unsupervised segmentation and reconstruction without the of pre-training. The procedure these steps: image over from quality segmentation based on unsupervised of for each identified by and based on these of segments based on for reconstruction and labeling in by a procedure to the volume. (b) segmentation of mouse SAC processes. of the dendritic in comparing and (d) Manual reconstruction of a amacrine cell and view (e) reconstruction of the same shown in and view Scale in b: 1 μm; 10 In we have a set of that rapid reconstruction of genetically identified neurons, so that their shapes and connections can be at high resolution in less time than required for conventional imaging and segmentation By the of neural ARTEMIS renders the interrogation of samples feasible and a to from neuronal to Materials and methods were used in with and by and at JAM-B-creER mice et al., generated in our mice (Rossi et al., and Cart-cre mice et al., were from In the the Cre recombinase gene was targeted to the endogenous this line expresses Cre in amacrine cells In the Cart-cre transgenic Cre expression is by elements from the In this Cre selective retinal ganglion cells. were on a and mice were used in this were to 100 at the time of were on standard The (Joesch et al., 2008) was provided by and the et al., line was generated and provided by and and a protocol The endoplasmic HRP (erHRP) was as First, to improve expression in the sequence of HRP peroxidase from a was to protein a signal sequence from the as well as an endoplasmic reticulum signal were at the and respectively. Finally, the was to and to H2O2 et al., The sequence of this is from peroxidase from (Martell et al., and the enhanced APEX2 from et al., were for expression in In the nuclear export signal was to The erHRP, APX, and APEX2NES were transfected to cells using the the were into a a Cre-dependent (AAV) with the and the et al., 2008) were generated by these together with and using a chemical method et al., the viral vectors be to in this are from gene a protocol For gene were with by A 30 was used to make a small in the the 1 of was and then 1 of or Cre-dependent of × per was injected into the with a and were and retinas were weeks following a protocol retinas were from in with at were made to the with ganglion cells up onto The tissue was with and by in then HEK-cells were with and for by a in were and in A small was made on the of the and the was from the This that the and staining could into the brain while the of the brain tissue during were with and for by a in cells and tissues were reacted with DAB to reveal of peroxidase activity, reduced with 50 mM sodium and stained with osmium. osmium tetroxide only in HEK cell of Figure 1—figure supplement or the reduced osmium and et al., et al., protocol other to the two consecutive the protocol the signal of membranes compared to the osmium. This reasonable at high an essential for our approach. Finally, the stained tissue was and with