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. 2021 Jul 22;10:e65445. doi: 10.7554/eLife.65445

Figure 1. Comprehensive molecular profiling of olfactory bulb projection neurons.

(A) Schematic representation of experimental design. Top: after injection of rAAVretro-CAG-H2B-GFP into PCx and AON, single nuclei were dissociated from three mice (single nuclei (sn) R1,2,3: replicates 1,2,3) and sorted using fluorescence-activated nuclei sorting (FANS). The population of nuclei is selected based on GFP and DRAQ5 (far-red fluorescent DNA dye). See Figure 1—figure supplement 2 for detailed FANS plots. Sorted nuclei were sequenced using 10x single-nucleus RNA-seq. Middle and bottom: after injection of rAAVretro-CAG-H2B-GFP into PCx (middle) or AON (bottom), single nuclei were dissociated from three mice for each injection site and sorted using FANS (as described above and Figure 1—figure supplement 2). RNA extracted from sorted nuclei was prepared and sequenced using bulk RNA deep sequencing. PCx: Piriform Cortex; AON: Anterior Olfactory Nucleus; R: replicate; GRN: Gene Regulatory Network. (B) Representative coronal sections and high-magnification images showing GFP expression (in green) in the main olfactory bulb after injection of rAAVretro-CAG-H2B-GFP into PCx and AON (top), PCx only (middle), and AON only (bottom). Injection of the virus into PCx and AON resulted in GFP-expressing nuclei located in the mitral cell (empty arrowheads), external plexiform, glomerular (white arrowheads), and granule cell layers; injection into PCx resulted in GFP-expressing nuclei located in the mitral cell layer (empty arrowheads); injection into AON resulted in GFP-expressing nuclei located in the external plexiform and glomerular layers (white arrowheads) and granule cell layers. GL: glomerular layer; EPL: external plexiform layer; MCL: mitral cell layer; GCL: granule cell layer. Neurotrace counterstain in blue. Scale bars, 100 μm and 50 μm (high magnification).

Figure 1.

Figure 1—figure supplement 1. Schematic representation of olfactory bulb cell types and their cortical projection targets.

Figure 1—figure supplement 1.

(A) Schematic representation of cell types and their distribution within the olfactory bulb (IN: interneuron, TC: tufted cell, MC: mitral cell, GL: glomerular layer, EPL: external plexiform layer, MCL: mitral cell layer, GCL: granule cell layer). Tufted and mitral cells project their axons to downstream cortical regions. (B) Schematic representation of the distinct axonal projection targets for mitral and tufted cells in the olfactory cortex (AON: anterior olfactory nucleus, PCx: piriform cortex). Tufted cells project primarily to AON and to the anterior part of PCx, whereas mitral cell axons target AON, the anterior and posterior portions of PCx.
Figure 1—figure supplement 2. Enrichment of GFP-expressing nuclei using fluorescence-activated nuclei sorting (FANS).

Figure 1—figure supplement 2.

(A) Representative FANS data of GFP-expressing nuclei after injection of rAAVretro-CAG-H2B-GFP into AON and PCx to label OB projection neurons. From left to right: gating strategy for enrichment of GFP-expressing nuclei. Nuclei are identified based on size and granularity in the FSC (forward scatter) versus SSC (side scatter) plot. Next, the population of nuclei restricted to Quadrant two is selected that is both GFP-expressing and DRAQ5-positive (far-red fluorescent DNA dye). To exclude doublets, the population of nuclei around the diagonal in the FSC-A (forward scatter area) versus FSC-H (forward scatter height) plot is selected. Lastly, GFP-expressing nuclei filtered through the preceding steps are plotted against the SSC-A (side scatter area). (B) FANS results for control specimen for AON and PCx (olfactory bulb contralateral to the injection hemisphere from the same animal) showing the absence of GFP-expressing nuclei (same gating strategy as shown in (A)). (C) Representative FANS data after injection of rAAVretro-CAG-H2B-GFP into PCx to enrich for mitral cell nuclei. (D) FANS results for control specimen for PCx injections showing the absence of GFP-expressing nuclei (same gating strategy as shown in (C)). (E) Same as in (C) but after injection of rAAVretro-CAG-H2B-GFP into AON to enrich for tufted cell nuclei. (F) FANS results for control specimen for AON (olfactory bulb contralateral to the injection hemisphere from the same animal) showing the absence of GFP-expressing nuclei (same gating strategy as shown in (E)).