Project description:To characterize the molecular diversity of olfactory bulb projection neurons we used viral targeting and Fluorescence Activated Nuclei Sorting (FANS) to enrich for olfactory bulb projection neurons, and single-nuclei RNA sequencing (sn-RNA seq) to comprehensively characterize their transcriptomes. To isolate GFP-labelled nuclei, 3 individual replicates of AON and PCx-injected mice were used. Ipsilateral and controlateral sides were minced separately and placed into two different tubes. The minced tissue was gently homogenized in Nuclei PURE Lysis Buffer and 10% Triton X-100 using an ice-cold dounce and pestle, and filtered two times through a 40 μm cell strainer on ice. After centrifuging at 500 rpm for 5 min at 4 °C, the supernatant was aspirated and gently resuspended in 500 μl of cold buffer (1x of cold Hanks' Balanced Salt Solution HBSS, 1% nuclease-free BSA, RNasin Plus and 1/2000 DRAQ5). Our study identifies molecularly distinct subtypes of mitral and tufted cells.
Project description:Transmission of olfactory information to higher brain regions is mediated by olfactory bulb (OB) projection neurons, the mitral and tufted cells. Although mitral/tufted cells are often characterized as the OB counterpart of cortical projection neurons (also known as pyramidal neurons), they possess several unique morphological characteristics and project specifically to the olfactory cortices. Moreover, the molecular networks contributing to the generation of mitral/tufted cells during development are largely unknown. To understand the developmental patterns of gene expression in mitral/tufted cells in the OB, we performed transcriptome analyses targeting purified OB projection neurons at different developmental time points with next-generation RNA sequencing (RNA-seq). Through these analyses, we found 1202 protein-coding genes that are temporally differentially-regulated in developing projection neurons. Among them, 388 genes temporally changed their expression level only in projection neurons. The data provide useful resource to study the molecular mechanisms regulating development of mitral/tufted cells. We further compared the gene expression profiles of developing mitral/tufted cells with those of three cortical projection neuron subtypes, subcerebral projection neurons, corticothalamic projection neurons, and callosal projection neurons, and found that the molecular signature of developing olfactory projection neuron bears resemblance to that of subcerebral neurons. We also identified 3422 events that change the ratio of splicing isoforms in mitral/tufted cells during maturation. Interestingly, several genes expressed a novel isoform not previously reported. These results provide us with a broad perspective of the molecular networks underlying the development of OB projection neurons.
Project description:Mitral and tufted cells are the projection neurons of the olfactory bulb (OB). We previously reported that somata location and innervation patterns were different between early- and late-born mitral cells (Imamura et al., 2011). Here, we introduced a plasmid that drives the expression of a GFP gene into the mouse OB using in utero electroporation, and demonstrated that we can deliver the plasmid vectors into distinct subsets of OB projection neurons by changing the timing of electroporation after fertilisation. The electroporation performed at embryonic day (E)10 preferentially labeled mitral cells in the accessory OB and main OB mitral cells in dorsomedial mitral cell layer (MCL). In contrast, the E12 electroporation introduced the plasmid vectors preferentially into main OB mitral cells in the ventrolateral MCL and tufted cells. Combining these data with BrdU injections, we confirmed that E10 and E12 electroporation preferentially labeled early- and late-born projection neurons, respectively. This work introduces a novel method for segregated labeling of mouse olfactory bulb projection neurons based on their birthdates. With this technique we found that early- and late-born projection neurons extend their secondary dendrites in the deep and superficial external plexiform layer (EPL), respectively. Although a similar segregation has been suggested for mitral vs. tufted cell dendrites, we found mitral cells projecting secondary dendrites into the superficial EPL in E12-electroporated main OB. Our observations indicate that timing of neurogenesis regulates not only somata location and innervation patterns but also the laminar organisation of projection neuron dendrites in the EPL.
Project description:Chronic olfactory inflammation (COI) in conditions such as chronic rhinosinusitis significantly impairs the functional and anatomical components of the olfactory system. COI induced by intranasal administration of lipopolysaccharide (LPS) results in atrophy, gliosis, and pro-inflammatory cytokine production in the olfactory bulb (OB). Although chronic rhinosinusitis patients have smaller OBs, the consequences of olfactory inflammation on OB neurons are largely unknown. In this study, we investigated the neurological consequences of COI on OB projection neurons, mitral cells (MCs) and tufted cells (TCs). To induce COI, we performed unilateral intranasal administration of LPS to mice for 4 and 10 weeks. Effects of COI on the OB were examined using RNA-sequencing approaches and immunohistochemical analyses. We found that repeated LPS administration upregulated immune-related biological pathways in the OB after 4 weeks. We also determined that the length of TC lateral dendrites in the OB significantly decreased after 10 weeks of COI. The axon initial segment of TCs decreased in number and in length after 10 weeks of COI. The lateral dendrites and axon initial segments of MCs, however, were largely unaffected. In addition, dendritic arborization and AIS reconstruction both took place following a 10-week recovery period. Our findings suggest that olfactory inflammation specifically affects TCs and their integrated circuitry, whereas MCs are potentially protected from this condition. This data demonstrates unique characteristics of the OBs ability to undergo neuroplastic changes in response to stress.