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

All experimental protocols were approved by the University of Texas Medical School at Houston and Yale University Institutional Animal Care and Use Committee in accordance with the National Institutes of Health guidelines. A total of 19 mice (11 OMP-Synapto-pHluorin knock-in mice, Jackson Lab, and 8 C57BL/6, Charles River) were anesthetized with nembutal (i.p. 50 mg/kg body weight). The animals were kept on a heating pad with circulating water set at 40°C. The bone covering the dorsal OB was carefully thinned, and a small opening was made (200–500 μm diameter) for glass pipette penetration. Dextran-conjugated Alexa 488 or Alexa 594 (Invitrogen) was diluted in Ca 2+ -free Ringer solution (140 mM NaCl, 5 mM KCl, 3 mM MgCl, 0.01 mM EDTA, 10 mM HEPES, and 10 mM glucose, pH 7.5) and used as 10% dye solution. Glomeruli were visualized by two-photon fluorescence imaging of OMP-SpH knock-in mice (Bozza et al., 2004 ). A dye-filled glass pipette (tip I.D., 2.5 μm) was placed into the target glomerulus under visualization with a two-photon microscope, or into the superficial or deep external plexiform layer (EPL) according to micromanipulator penetration depth (100–150 or 200–250 μm depth, respectively). Small square-wave pulses were delivered via the pipette for dye electroporation (3–5 μA, 25-ms pulse width, 2 Hz, 1200–3600 pulses). The electroporation effective area using this technique has been previously reported to be about 30 μm in diameter (Nagayama et al., 2007 ), and was effective to label a single glomerulus or superficial/deep EPL in the OB. Three to 5 days after the dye injection, animals were deeply anesthetized by nembutal injection (i.p. 150 mg/kg) and perfused transcardially with 0.9% NaCl followed by 4% paraformaldehyde in 0.1 M phosphate buffer. Brains were carefully withdrawn from the skull, and the ventrolateral aspect of the whole brain was imaged using a custom-built two-photon microscope system. The imaging system consisted of a mode-locked laser, operating at a 100-fs pulse width, 80-MHz pulse frequency, and 810-nm wavelength (Tsunami Millennia Xs or Mai Tai DeepSee, Spectra Physics), and an Olympus Fluoview scan box mounted on an upright BX50WI microscope. Images were captured with a 10× Olympus water immersion-type objective lens (NA = 0.3). Images were collected at different focus planes with a 10-μm interval. The images were stacked and tiled for enlarged views using Image-J and Photoshop software. Some brains were post-fixed overnight and sectioned with a vibratome to observe the dye injection site. The 50-μm sections were mounted on glass slides using mounting medium that included DAPI (Vectashield, VECTOR), and images were captured using an Olympus microscope with a CCD camera (Sensicam). In total, single-glomerulus dye injections were carried out in six brain hemispheres, and six superficial/deep EPL dye injection samples were used in this study for data analysis.

Limitations of the tracing method

To interpret the data accurately, the physical drawbacks of this tracing method must first be discussed. This method has the following difficulties and limitations: (1) tracing of obscured brain surface areas, such as the rhinal sulcus and septal regions of the OC, lateral entorhinal cortex, and amygdaloid cortex; (2) detection of weakly labeled and small branches; (3) detection of the axons that extend into deeper brain areas such as the ventrorostral anterior PC (APC VR ) region beneath the LOT (Ekstrand et al., 2001 ); and (4) identification of individual axons in axon-congested areas such as the anterior LOT in the area of the AOC. Comparatively, although classical single-neuron approaches have a higher quality of individual axon tracing in regard to the limitations above, our method allows the tracing and comparison of multiple axons simultaneously within the same sample. This advantage is critical for the exploration of large neuroanatomical circuits and allowed us to compare the individual axon trajectories arising from a common glomerulus as well as from differentially labeled presumed mitral and tufted cell populations. Further use of this method could reveal OB–OC connections more clearly and completely in future investigations.