Differential axonal projection of mitral and tufted cells in the mouse main olfactory system

Abstract

In the past decade, much has been elucidated regarding the functional organization of the axonal connection of olfactory sensory neurons to olfactory bulb (OB) glomeruli. However, the manner in which projection neurons of the OB process odorant input and send this information to higher brain centers remains unclear. Here, we report long-range, large-scale tracing of the axonal projection patterns of OB neurons using two-photon microscopy. Tracer injection into a single glomerulus demonstrated widely distributed mitral/tufted cell axonal projections on the lateroventral surface of the mouse brain, including the anterior/posterior piriform cortex (PC) and olfactory tubercle (OT). We noted two distinct groups of labeled axons: PC-orienting axons and OT-orienting axons. Each group occupied distinct parts of the lateral olfactory tract. PC-orienting axons projected axon collaterals to a wide area of the PC but only a few collaterals to the OT. OT-orienting axons densely projected axon collaterals primarily to the anterolateral OT (alOT). Different colored dye injections into the superficial and deep portions of the OB external plexiform layer revealed that the PC-orienting axon populations originated in presumed mitral cells and the OT-orienting axons in presumed tufted cells. These data suggest that although mitral and tufted cells receive similar odor signals from a shared glomerulus, they process the odor information in different ways and send their output to different higher brain centers via the PC and alOT.

Keywords: axon projection; mitral cell; olfactory bulb; olfactory tubercle; parallel pathways; piriform cortex; tufted cell; two-photon imaging.

Figure 1

Axonal trajectories of neurons from a single glomerulus. (A) Two-photon images of the ventrolateral aspect of the whole brain. A schematic diagram of the dye injection and observation areas from the same ventrolateral view of the whole brain is shown in the upper left. Inset: One-shot image of the dye injection pipette and the target glomerulus before dye injection. Main figure: Two-photon images of the olfactory cortex in the same animal. Labeled axons appeared on the brain surface near the border between OB and AOC (upper left part of the image) and tracked along the LOT and branch into a/pPC and OT. An area of dense axonal projection appears in the anterior OT (yellow dotted circle). Non-neuronal cells on the surface of the brain also uptake dye and show spotty noise in the image. AOC, anterior olfactory cortex; aPC, anterior piriform cortex; pPC, posterior piriform cortex; OT, olfactory tubercle. (B–G) Highlights of the individual axon trajectories in (A). (H) Superimposition of the images (A–G). PC-oriented axons mainly run along the dorsal part of the LOT, while OT-oriented axons mainly run along the ventral part of the LOT. The population of the single-glomerulus axons covers a wide area of the OC. Presumed cortical borders are drawn in yellow. (I) Axon projection orientation index. Nineteen axons were categorized into five subgroups on the basis of their axon projection orientation index calculated as (OT projection branches)/(OT + PC branches). PC, OT, and neutral projection groups are shown as green, red, and gray columns, respectively.

Figure 2

Superficial and deep EPL cell axon projection pattern. (A) Inset: Coronal image of the tracer injection site in the OB. Dextran-conjugated Alexa 488 (green) and Alexa 594 (red) were injected into the deep and superficial parts of the EPL, respectively, in the same area of the dorsal OB. The majority of the red-labeled neurons were tufted cells, and most green-labeled cells were mitral cells. DAPI nuclear labeling is blue. Main figure: Cortical observations in the same animal. The majority of green-labeled presumed mitral cell axons run along the dorsal part of the LOT and mainly branch into the a/pPC, although faintly green-labeled axons can also be observed in the OT. The majority of red-labeled presumed tufted cell axons run along the ventral part of the LOT and mainly project into the OT, although a small number of red-labeled axons can also be observed in the a/pPC. (B) Distribution of the labeled neurons in the OB. Neurons labeled by the superficial EPL (S-EPL; n = 37) and deep EPL (D-EPL; n = 82) dye injections were categorized in two groups, mitral cell layer cells (MCL cells; Green) and external plexiform layer/glomerular layer cells (EPL/GL cells; Red). (C) Axon projection orientation index. The majority of superficial EPL neurons (red column; n = 30) were grouped in the higher index number categories (right side of the graph), while deep EPL neurons (green columns; n = 34) tended to have lower index numbers (left side of the graph).