1. Volume 2, Issue 5, Pages 1375-1386 (November 2012)
Charting Monosynaptic Connectivity Maps by Two-Color Light-Sheet Fluorescence Microscopy 
Christian J. Niedworok, Inna Schwarz, Julia Ledderose, Günter Giese, Karl-Klaus Conzelmann, Martin K. Schwarz 
Cell Reports 
Volume 2, Issue 5, Pages (November 2012)
DOI: /j.celrep
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2. Cell Reports 2012 2, 1375-1386DOI: (10.1016/j.celrep.2012.10.008)
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3. Figure 1
Stereotaxic Virus Delivery into the Olfactory Bulb of Adult Mice
(A) Schematic showing the different viruses used for stereotaxic injections. Two rAAVs subtype 8, one expressing tdTomato linked with an IRES to TVA, the other expressing rabies glycoprotein, both under control of a CBA promoter, are mixed with an EnvA pseudotyped, glycoprotein-gene deleted rabies virus expressing EGFP (RABVΔG-EGFP [EnvA]).
(B) Schematic showing the stereotaxic injection of the virus cocktail into the main OB of an adult mouse (p56) and the timeline from injection to analysis (bottom). The needle of a syringe (green: RABVΔG-EGFP [EnvA], red: rAAV mix) is inserted into the mouse brain (dashed red lines) at the level of the OB. Yellow dots represent the source cell population. Green dots indicate directly connected (red arrow) presynaptic neurons. See also Figure S1. rAAV, recombinant adenoassociated virus; CBA, chicken beta-actin promoter/enhancer; IRES, internal ribosomal entry site; tdTomato, tandem dimer Tomato red-fluorescent protein; RABVΔG-EGFP (EnvA), glycoprotein gene-deficient, EGFP expressing rabies virus encapsulated with the avian sarcoma and leukosis virus envelope protein EnvA; TVA, tumor virus A; p56, postnatal day 56.
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4. Figure 2 RABV Spread after Stereotaxic Delivery into the Main OB
(A) Confocal mosaic image of a coronal section from the OB of an adult mouse 8 days after virus-cocktail delivery. White dotted ellipse marks the point of virus delivery in the GCL. White arrows point at the different layers (GL, EPL, MCL) of the OB. Blue: DAPI fluorescence. Double-infected tdTomato and EGFP expressing neurons appear in yellow. Red neurons express tdTomato only. Green RABV-infected neurons express EGFP only.
(B) High magnification of double-infected neurons (exemplary white arrows) in the GCL from (A).
(C) Confocal mosaic image of a coronal section through the brain (Bregma 0.02 mm) of an adult mouse after virus-cocktail delivery into OB. PC (1) and HDB (2) are marked by white squares and contain numerous RABV-infected, EGFP-positive neurons.
(D and E) Closeup of (1) and (2) from (C). Note the high density of EGFP-positive neurons in HDB (E).
See also Figures S2, S3, and S4. Blue: DAPI fluorescence; OB, olfactory bulb; GCL, granule cell layer; GL, glomerular layer; EPL, external plexiform layer; MCL, mitral cell layer; CTX, cerebral cortex; CPu, caudate putamen; LV, lateral ventricle; HDB, horizontal limb of the diagonal band; PC, piriform cortex.
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5. Figure 3
TVA Receptor and Rabies Glycoprotein Dependency of Modified RABV Infection
(A) Fluorescence image of a 70 μm coronal section from the OB of an adult mouse injected with an EnvA pseudotyped, glycoprotein gene-deleted rabies virus expressing EGFP (RABVΔG-EGFP [EnvA]) (bottom). DAPI fluorescence appears in blue. White arrow from top points at stereotaxic injection site. White arrows from left point at different layers (GL, EPL, MCL) of the OB. No EGFP-positive cells were found.
(B) Fluorescence image of a 70 μm coronal section from the OB of an adult mouse injected with a virus cocktail containing an rAAV expressing the TVA receptor and the EnvA pseudotyped, glycoprotein gene-deleted rabies virus expressing EGFP (RABVΔG-EGFP [EnvA] [bottom]). The white arrow from the top points at the injection site containing numerous EGFP-labeled (green) neurons. The white arrows from the left point at different layers of the OB.
(C) Fluorescence image of a 70 μm coronal section through the brain (Bregma 0.02 mm) of an adult mouse injected into the OB with a virus cocktail containing an rAAV expressing the TVA receptor and the EnvA pseudotyped, glycoprotein-deleted, RABV-expressing EGFP (RABVΔG-EGFP [EnvA]).
Blue: DAPI fluorescence. HDB and PC are encircled (white dotted lines). No EGFP-positive (green) neurons can be detected.
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6. Figure 4
Preservation of EGFP and mRFP1 Fluorescence after Optical Clearing and Light-Sheet Fluorescence Microscopy Principle
(A) Picture of a perfused mouse brain 8 days after stereotaxic injection of rAAV-CBA-EGFP IRES TVA in the right, and rAAV-CBA-mRFP1 IRES TVA the left OB (injection volume: 300 nl each, Bregma: 4.28 mm, lateral: 0.5 mm, ventral: 2.5 mm).
(B) Picture of the same brain after optical clearing for light-sheet microscopy.
(C) Uncleared brain revealing bright EGFP fluorescence (green) in right OB.
(D) Cleared brain revealing preservation of EGFP fluorescence (green) in right OB.
(E) Uncleared brain revealing bright mRFP1 fluorescence (red) in left OB.
(F) Cleared brain revealing bright mRFP1 fluorescence (red) in left OB.
(G) Schematic illustrating the principle of light-sheet fluorescence microscopic imaging. The cleared mouse brain is placed under a detection objective and illuminated from one side with the light sheet (blue) generated by scanning through an illumination objective. The brain is moved in x, y, z directions to generate a virtual mosaic grid (red).
(A)–(F) Images taken with a dissecting microscope in trans-/epi-illumination (A), transillumination (B), green fluorescence (C and D), or red fluorescence mode (E and F). See also Movie S1. EGFP, enhanced green fluorescent protein; mRFP1, monomeric red fluorescent protein 1.
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7. Figure 5
Light-Sheet Fluorescence Microscopic Images of RABV-Labeled Neuronal Circuits
(A–C) Maximum intensity projections of an entire mouse brain generated from a single horizontal LSFM scanning session after virus-cocktail injection into AON (0.8× detection lens; three channels recorded, only EGFP shown; x/y: 6.7 μm/pixel; z-step: 10 μm; 451 z-steps; recorded volume 9.2 × 6.9 × 4.5 mm; time 36 min; size: 3.7 bit). RABV-infected, EGFP-positive neurons can be detected in OB and in several prominent cortical and subcortical structures (AON, PC, HP). (A) Virtual sagittal view. (B) Horizontal view. The white dotted square marks the region showing the visualization of axon bundles from AC (white arrow points at AC). (C) Virtual coronal view.
(D) Magnification of AC marked in (B). Note that the brightness was increased to improve the ACs visibility. White arrows point at the axon-bundles of AC connecting the ipsi- and contralateral AONs. CB, cerebellum; AON, anterior olfactory nucleus; AC, anterior commissure; HP, hippocampus; DBB, diagonal band of Broca.
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8. Figure 6
Light-Sheet Fluorescence Microscopic Imaging of Neuronal Populations Monosynaptically Connected with GCs or PGCs
(A–H) Maximum intensity projections generated from two horizontal LSFM mosaic scanning sessions (10× lens; two channels; x/y: 639 nm/px; z-step: 3 μm; A–D: 679 z-steps; 7 × 8 tiles; volume ∼6.0 × 5.0 × 2.0 mm; E–H: 761 z-steps; 9 × 5 tiles; volume ∼7.1 × 2.9 × 2.3 mm). Z ranges are measured from the highest point on the surface of the brain in the displayed area along the z axis of the viewpoint.
(A) Overview of OB and rostral forebrain. Red rAAV-infected neurons are confined to the GCL of the OB. Yellow-labeled neurons in the center of the OB represent rAAV and RABV-infected postsynaptic source neurons. Green EGFP-positive neurons in OB, PC, and DBB including HDB represent RABV-labeled presynaptic neurons, monosynaptically connected to the source cell population in OB. A red artifact is visible on the right. White squares mark the regions enlarged in (B–D).
(B) Optical section through the GCL. White arrows point at red (rAAV-infected), green (RABV-infected), and yellow (coinfected) GCs.
(C) Optical section (EGFP channel) through the PC revealing bright fluorescent neurons.
(D) Optical section (EGFP channel) through HDB revealing numerous brightly fluorescent neurons.
(E) Overview of OB and rostral forebrain. rAAV-positive neurons (red) are visible in the GL of the OB. rAAV and RABV double-infected postsynaptic source neurons (yellow) can be detected in the GL of the OB. RABV-infected neurons (green) monosynaptically connected to the source cell population can be found in OB, PC, and DBB (white arrows) including HDB. White squares mark the regions enlarged in (F–H).
(F) Optical section through the GCL of the OB. White arrows point at rAAV-infected, RABV-infected and coinfected PGCs.
(G) Optical section (EGFP channel) through the PC revealing bright fluorescent neurons.
(H) Optical section (EGFP channel) through HDB revealing numerous brightly fluorescent neurons.
See also Figures S5, S6, and S7 and Movie S2. GCs, granule cells; PGCs, periglomerular cells.
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9. Figure 7
Preservation of EGFP Fluorescence and Antibody Staining after LSFM Imaging, Rehydratation, and Sectioning
(A) Confocal image of a 70 μm vibratome section after LSFM imaging and rehydratation showing RABV-EGFP-labeled neurons in HDB.
(B) Same section as in (A) stained against acetocholin (Ach) revealing cholinergic neurons in HDB (red).
(C) Overlay of (A) and (B) identifying cholinergic nature of RABV-labeled neurons (white rectangle).
(D–F) Magnification of the region shown in (C, white rectangle) showing a RABV-EGFP-labeled, cholinergic neuron (white arrow). See also Figure S6.
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10. Figure S1
Efficient Double Infection of Individual Neurons by Two Different rAAVs, Related to Figure 1
Epifluorescence images of a 70 μm coronal section from the OB of an adult mouse injected with a 1:1 mixture of rAAVs subtype 8 expressing either EGFP or tdTomato under the control of a CBA promoter (bottom left). (A) Green fluorescence revealing expression of EGFP. (B) Red fluorescence revealing tdTomato. (C) Merged image showing double-infected neurons (yellow) in all layers of the olfactory bulb around the injection site. Blue: DAPI fluorescence. Arrow from the top points at the injection site, small arrows in the enlarged inset (rotated 90° counterclockwise) point at exemplary double-positive cells. OB, olfactory bulb; GCL, granule cell layer; GL, glomerular layer; EPL, external plexiform layer; MCL, mitral cell layer.
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11. Figure S2
Axonal Projections into Main Olfactory Bulb from Piriform Cortex and the Horizontal Diagonal Band, Related to Figure 2
(A–D) Confocal image of a horizontal section through the OB and the anterior part of the prefrontal cortex of an adult mouse 3 weeks after stereotaxic rAAV-EGFP injection into the PC and the HDB. Axons with strong EGFP labeling (green) are visible in the GCL and the GL of the OB. Note that the AOB is virtually devoid of EGFP-positive axons. (B) Magnification of inset 1 from (A). White arrowheads point at intensely EGFP-labeled axons in the granule cell layer. (C) Magnification of inset 2 from (A). White arrowheads point at exemplary EGFP-labeled axons in the GL. (D) Schematic showing the injection sites in PC and the HDB. AOB, accessory olfactory bulb; EPL, external plexiform layer; GCL, granule cell layer; GL, glomerular layer; MCL, mitral cell layer; PC, piriform cortex; HDB, horizontal diagonal band; A, anterior; L, lateral.
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12. Figure S3
Modified RABV-Mediated Transsynaptic Tracing of GC, Related to Figure 2
(A) Confocal mosaic image (EGFP channel) of a coronal section through the OB of an adult mouse 7 days after stereotaxic delivery of the tracer virus cocktail (rAAVs/modified RABV) into the GCL. The syringe containing the virus cocktail (green) points at the injection site. Bright source GCs can be detected in the GCL at the injection site. Directly connected, presynaptic neurons are confined to the EPL. Note the absence of EGFP-labeled MCs in the MCL, suggesting that the RABV cannot cross dendrodendritic, reciprocal synapses.
(B) Magnification of the region in (A) outlined by the white dotted rectangle demonstrating the absence of RABV-labeled, EGFP-positive MCs. White dotted lines demarcate the EPL. OB, olfactory bulb; GCL, granule cell layer; MCL, mitral cell layer; EPL, external plexiform layer; GL, glomerular layer.
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13. Figure S4
Cell Death after Prolonged Virus Cocktail Exposure, Related to Figure 2
Confocal image of a 70 μm coronal section through the OB 15 days after virus cocktail injection into the GCL (syringe) showing the appearance of GCs with distorted morphology as well as EGFP-positive glia cells (white arrows, inset). OB, olfactory bulb; GCL, granule cell layer; EPL, external plexiform layer.
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14. Figure S5
Virus-Mediated Labeling of Periglomerular Source Cells and Monosynaptically Connected Neurons in Olfactory Bulb and Nasal Epithelium, Related to Figure 6
(A) Confocal image of a coronal section through the OB of an adult mouse 7 days after stereotaxic delivery between dura (red line) and GL of the tracer virus cocktail (rAAVs/modified RABV). Inset shows a magnification of a typical monosynaptically connected interneuron in the internal plexiform layer of the OB.
(B) Confocal image of a coronal section through the rostral mouse head at the level of the OB showing the efficient transsynaptic labeling of OSNs (green EGFP fluorescence) in the nasal epithelium (NE) after primary infection of periglomerular cells with the virus cocktail. White square marks a part of the NE shown at higher magnification in the bottom left corner. EGFP-positive OSNs light up in green. GL, glomerular layer; EPL, external plexiform layer; MCL, mitral cell layer; GCL, granule cell layer; NE, nasal epithelium; OSN, olfactory sensory neurons.
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15. Figure S6
Consecutive Optical Coronal Sections after Virus Injection into the GL, Related to Figures 6 and 7
Consecutive optical sections (EGFP channel, 300 μm each) through OB and ventral forebrain after LSFM imaging showing the neurons in PC and DBB directly projecting onto interneurons in the PGCL. White arrows point at injection site (is) and periglomerular cell layer (PGCL), respectively. Note the densely packed neurons in HDB. OB, olfactory bulb; is, injection site; PGCL, periglomerular cell layer; PC, piriform cortex; DBB, diagonal band of Broca; HDB, horizontal limb of the diagonal band.
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16. Figure S7
RABV-Mediated Transsynaptic Tracing of Presynaptic Inputs onto Single MCs, Related to Figure 6
(A) Schematic of the head of a newborn (P0) mouse injected bilaterally into the lateral ventricles with an rAAV expressing RG under control of the synapsin promoter (rAAV8 syn-RABVGlyco). Injection results in selective labeling of MCs in the OB (left).
(B) Schematic of a sagittal section through an adult mouse brain injected at P0 with rAAV8 syn-RABVGlyco and subsequently injected into the PC with a RABV expressing EGFP (RABVΔG-EGFP) (green syringe). Triangles in OB represent MCs. Green dots in OB represent monosynaptically connected interneurons in EPL. Green lines indicate MC axons projecting into PC (green ellipse).
(C) Confocal image of a RABV-infected, rAAV8 syn-RABVGlyco expressing MC (EGFP channel) showing monosynaptically connected interneurons in EPL. White arrow points at MCs. White ellipses indicate the position of glomeruli.
(D) Another example of single MC connectivity maps as described in (B).
Note that labeling of GC is absent, suggesting that the RABV cannot cross dendrodendritic, reciprocal synapses. OB, olfactory bulb; CTX, cerebral cortex; HP, hippocampus; PC, piriform cortex; MC, mitral cells; MCL, mitral cell layer; EPL, external plexiform layer; GL, glomerular layer.
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