Introduction

In mammalian brains, neural precursor cells originate from the subventricular zone (SVZ) and give rise to neuronal precursor cells that migrate to the olfactory bulb (OB). In the OB, these cells differentiate into new neurons, which are thought to be involved with olfactory processing and learning. Over the past decade, magnetic resonance imaging (MRI) has been demonstrated to be able to detect single cells and to track new neurons in the OB of live animals [1,2,3]. This requires labeling the precursor cells with iron oxide based contrast to give sufficient MRI signal. Neural progenitor cells in the SVZ were labeled with micron-sized iron oxide particles (MPIOs). Following their migration into the OB, the distribution of MPIO-containing new neurons in the OB were detected with high resolution MRI (Figure 1). In this study, we aimed to confirm that the MRI signals could be used to detected single new neuron in the OB and whether the distribution of new neurons could be mapped in laminar specific manner. Finally, we investigate whether odor enrichment can influence the spatial distribution of new neurons in the OB of live animals.

Method

Identification of MPIO within single new neuron was analyzed on OB tissue sections. Adult male rats (n =4) 6 weeks of age received intraventricular injection of MPIOs (Bangs Laboratories, Inc., Fishers, IN) as previously described [3]. 4 weeks after injection, the animals were euthanized and perfused with 5% formalin and the OB were removed and processed for cryosection to obtained thin OB section (25 micron-thick). Total of 8 OB sections from each rat were randomly chosen and processed for ex vivo MRI and immunohistochemistry analyses (Figure 2). For in vivo experiment, a separate group of the rats (n =3) were injected with MPIOs and kept for 4 weeks. 24 prior to MR imaging, animals were infused with 37mg Mn²⁺/kg. Data was acquired on an 11.7 T animal MRI system (30 cm 11.7 T horizontal magnet, (Magnex Scientific, Oxford, England), MRI Electronics, Bruker Biospin, Billerica, MA, and 12 cm 3D gradients (Resonance Research Inc, Billerica, MA) using a volume transmit coil and a custom built, 1 cm diameter, receive-only surface-coil. Animals were imaged at four weeks post-injection of MPIO and were placed in a MRI compatible cradle with a stereotactic head-frame under continual anesthesia of 2% isoflurane in 75% O2/25% medical air. Animals were orally intubated and mechanically ventilated at 50-60 breaths/min while the end tidal CO2 and respiration patterns were monitored. Body temperature was maintained at 37° C using a circulating water bath. 3D Multi-gradient echo (MGE) sequences were used for MRI with the following parameters: FOV 1.28 cm x 1.44 cm x 0.96 cm, Matrix 256 x 288 x 192 (50 mm isotropic resolution), 50 kHz bandwidth, multiple TEs 4.25, 11.75, 19.25, and 26.75 ms, and TR 32 ms. Images were reconstructed using MIPAV. Images from the second and third echo were thresholded at 3 x standard deviation of the noise of the surrounding 5 pixels to select the MPIOs from the background. This thresholded mask was overlaid on the original images from the first echo. From these composite images, the MPIOs were counted in each layer of the olfactory bulb

Results and Discussion

In this study, we demonstrate that MRI can be used to identify single new neurons in the rat olfactory bulb (OB) with layer specificity. The MPIO-labeled neuroblasts give a strong T2* effect and can be easily identified as hypointense spots. Moreover, signals measured from MRI were well-correlated with MPIOs (Figure 3). Immunohistological identification confirmed that the MPIOs were indeed maintained in the new neurons following their differentiation from precursor cells and had limited non-specific uptake in astrocytes and microglia (Figure 4). Therefore, the results show that MPIO-generated MRI contrast can be used to detect individual labeled new neurons and their integration within the OB. Furthermore, when combining this method with Mn-enhanced MRI (MEMRI), it could be applied to study the effect of odor enrichment on the layer specific distribution of the new neurons in the OB. The MPIO-positive cells were imaged in the olfactory bulb with the aid of Mn²⁺ (Figure 5). The addition of manganese was useful not only because it is excellent T1 relaxation agent, which increases the signal to noise, but also has the unique property of enhancing cytoarhitecture of the olfactory bulb [4]. The ability to map distribution of the new neurons in vivo using MRI should afford interesting new avenues for future study with regard to adult neurogenesis and experience-dependent integration of new neurons.

Acknowledgements

This research was supported by the NINDS Intramural Research Program of NIH.

References

[1] Shapiro, E.M., Gonzalez-Perez, O., Manuel Garcia-Verdugo et al., 2006, Neuroimage 32, 1150-1157. [2] Sumner, J.P., Shapiro, E.M., Maric, D. et al., 2009, Neuroimage 44, 671-678. [3] Pothayee N., Cummings D.M., Schoenfeld T. et al., 2017, Neuroimage, 158., 232-241. [4] Aoki, I., Wu, Y.J., Silva, A.C.et al., 2004, Neuroimage 22, 1046-1059.